AU2014275166B2 - Compositions and methods for identification, assessment prevention, and treatment of cancer using PD-L1 isoforms - Google Patents

Abstract

The present invention relates to methods for identifying, assessing, preventing, and treating cancer (

Description

COMPOSITIONS AND METHODS FOR IDENTIFICATION, ASSESSMENT, PREVENTION, AND TREATMENT OF CANCER USING PD-Li ISOFORMS

Cross-Reference to Related Apolications Thisapplication claims the benefit ofU S. Provisional Application No. 61 /831.894, filed on 6 June 2013; theentire contents of said application is incorporated herein in its entirety by this reference.

Statement of Rights This invention was made with government support under Grant NIH U24CA143867 awarded by the National Institutesof Health. The U.S. government has certainkrghts in the invention. This statement is included solely to comply with 37 C.FR. § 401,14(a)(f)(4) and should not be taken as an assertion or admission that the application discloses and/or claims only one Invention.

Background of the invention In order for T cells to respond to foreign proteins, twosignals must be provided by antigen-presenting cells (APCs) toresting'T lymphocytes (Jenkins, M and Schwartz, R. (1987)J pfap. e 165:302-319; Mueller, D.L, e ao (1990) JnnnunoL 144:3701-3709). Thefirstsignal whichconfers specificity totheimmune response, is transduced via the T celi recepior (TCR) following recognition of foreign antigenicpepidepresented in the context of the major histocompatibility complex (MHC) The second signal, termed costimulation, induces T cells to proliferateand become functional (Lenschow ita (1996) Annu. Rev. Inmunol 14:233). Costimulation is neither antigen-specific, nor MHC restrictedand is thought to be provided by one or more distinct cell surface polypeptides expressed by APCs (JenkimnsM. K. etal (1988)J imnunol 140:3324-3330; imnsley, P. et aL (1991)J xp. led 173: 721-7 30Girnni, C. D- e a 1991 Proc. NatL Aca Sci. 1SA 88:6575-6579, Young, J W. et a! (1992)J C/in. Invest 90:229-237; Koulova, L. t a. (1991)1 Exp. Md 173:759-762; Reiser, H. et ca (1992) Proc. Na. cad Sci tA 89:271-275; van-Seventer, G. A.et dat (1990).linmunot 144:4579-4586; LaSalle, J. M. et at (1991),inmno. 147:774-80: Dustin M. 1 el al (1989)1 1. ed 169:503; Armitage, R.I, et al (1992) Nature 357:80-82; Liu, Y. et aL (1992)J 1 .Med 175:437 445),

The CD80 (B7-1)and CD86 (B7-2) proteins, expressed on APCs, are critical costimulatory polypeptdes (Freeman e al 0(991) 1&ped 14:625; Freeman et al. (1989)3 ImmunoL 143:214Azumat (199)\Natre366'6Freemancet at(1993) Science 262:909). B7-2 appears to ply-a predominant role duringprimary immune responses, hileB7-1, which is upregulated later in the course of an immune response, may be important in prolonging primary T cell responses or costirnulaiing secondary T cell responses (Bluestone (1995) hmnmity 2:555). One receptor to which B7-1 and B7-2 bind, CD28, is constitutively expressed on resting T cells and increases in expression after activation. After signaling through the T cell receptor, ligation of CD28 and transduction of a costimulatory signal inducescellsto proliferate and secrete R-2 (Linsley, P. S. et at (1991)./4 &ped 173:721~730; Gimmi, C. D, et al (1991) Proc. NaX Acad, Sci, USA 88:6575-6579, June, C, L e al. (O1990) 1mmuno. Todav. 11:211-6; Harding, A, et t (1992) Naure 356:607-609)- A second receptor, termed CTLA4 (CD152) is homologous to CD28 but is not expressed on resting T cells and appears following T cell activation (Brunet, J. F. et al (1987) Naure 328:267 270). CTLA4 appears to be critical in negative regulation of T cell resposes(Waterhouse e a.(1995) Scenc270:985).BlockadeofCTLA4 hasbeen foundtoremove inhibitory signals, while aggregation of CTLA.4 has been found to provide inhibitory signals that downregulate T cell responses (Allison and Krumnel (1995) Science 270:93,) ThcB7 polypeptideshave a higher affinity for CTLA4 than for CD28 (Linsley, P. S, et al (1991).. ExMed 174:561-569) and B7-1 and B7-2 have ben Found to bind to distinct regions of the CTLA4 polypeptide and have different kinetics of binding to CTLA4 (Linsley e al. (1994)]mmn/v[1:793).[COS, which isapolypeptide related toCD28 appears tobe important in IL-10 production (HutloffcetaL.(1999) Nare397:263;WO 98/38216),as has its ligand (Aiher A. e caL. (2000).immun164:4689~96; ages H.. W. et a (2000) Eur . hnmunoL 30:1040-7; Brodie D. et aL (2000) CIurr, iol 10:333-6; Ling V. et al. (200) IJhnmunoL 164:1653-7; Yoshinaga S.K. e at (1999) %Nature 402:827-32). If T cells are only estimated through the T cell receptor, without receiving an additional costimulatory

signal, they become nonresponsive, anergic, or die, resultingin dowmnodulation of the immune response. The importance ofthe B7:CD28/CTLA4 cosimulatory pathway has been demonstrated in vitroand in several in vivomodel systems, Bkockade ofthis cstimulatory pathway results in the development ofantigen specific tolerance in marine and human systems (HardingF.A. t(1992) Nature356:607-9; Lenschow, D, , et al (1992) Science 257:789-792; Turka, L. A. ela! (1992.) Proc Ntl Acad, Sci. USA 89:11102 1105; Ginni, C, D. et al (1993) Proc. NaL A cad. Sci. USA90:6586-6590; Boussiotis, V. et a, (1993)J, Ex. d. 178:1753-1763). Conversely, expression of B7 by B7 negative mnurine tumor cells induces Tcell mediated specific immunity accompanied by tumor rejection and long losing protection to rumor challenge (Chen, L, el al (1992) Cell 71:1093-1102; Townsed, S. E and Allson, J. P. (1993) Science259:368-370; Baskar, S. e ad. (1993) Proc. Nt A cad, Sci. 90:5687-5690.). Inhibitory receptors that bind to costimulatory polypeptideshave also been identified on immune cells. Activation ofCTLA4, for example, transmits a negative signal to aT cell EngaIgement ofCTLA4 inhibits I.-2 production and can induce cell cycle arrest (Krunimel and Allison (1996)J Erp Med, 183:2533), Inaddition, mice that lack CTLA4 developlyphoproliferative d1sase (ivil e a (1995)Immunitv3:541; Waterhouse et at (1995)Science270:985).The blockade of CTLA4 withantibodies may remove an inhibitorysignal, whereas agregation ofCTLA4 with antibody transmits aninhibitor signal Therefore, depending upon thw receptor to which a costimulatory polypepide binds (iAct a cosimulatory receptor such as CD28 or an inhibitory receptor such as CTLA4), certain B7 polypeptides can promoteT cell costimulation or inhibition. PD-1 has been identified as a receptor which binds to PD-Ll and PD-L2 PD-1 is a member of the immunoglobulngnLpe superfamily. PD-I (shida et al (1992)EMOC)J 11:3887; Shinohara et al (1994) Genomics 23:704; U.S. Patent 5,698,520) has an extracellular region containing immunoglobulin superfamily domain, a transmenmbrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif(ITIM), These features also defme a larger family ofpolypeptides, called the iimunoinhibitory receptors, whichalso includes gp49B, PlR-B, and the killer inhibitory receptors (KIRs) (Vivierand Dacron (1997) nnmunoL Todcy 18:286). It is often assumed that the tyrosyl phosphorylated [TIM motif of these receptors interacts with S2-domain containing phosphatases, which leads to inhibitory signals. A subset of these inmnmoinhibitory receptors bind to MHC polypeptids, for example the KIRs, and CTLA4 bind to 1B7-1 and B7-2, It has been proposed that there is a phylogentic relationship between the MHC and B7genes (Henry et a. (1999) immmol Todav 20(6285-8), The nucileotide and amino acid sequence of PD-i is published in Ishida et aL (1992) EMBOJ 11:3887; Shinohara e al. (1994) Genomics 23:704; and U.S. Patent 5,698,520.

PD-I was previously identified using subtraction cloning basedapproach to select for

proteins involved inapoptotic cell death. Like CTLA4, PD-i is rapidly induced on the surface of T-cells in response toanti-CD3 (Agat et al, (1996)hit. Immunol 8:765). In contrast to CTLA4. however, PD- is also induced on the surface of B-cells (in response to anti-lgM). PD-I is also expressed ona subset of hymocytcs and mycloid cells (Agata elad (1996) supra; Nishimura el a/ (1996) In.]nmiol. 8: 773). Two types of human PD-I ligand polypeptides have been identified. PD-] ligand proteins comprise a signal sequence, and anigV domain, an IgC domain, a transmembrane domain, and a short cytoplasmic tail. Both PD-L (Freeman at(2000) JExp.Med. 192:1027) awdPD-L2 (Latchman el al (2001) Namnnmol 2:261) are members of the B7 family of polypeptides. Both PD-1 Iand.PD-L2 are expressed in placenta,spleen, lymph nodes, thymus and heart. Only PD-L2 is expressed in pancreas, lung and liver while only PD-LI ixprssed infetalliver.Both PD-1 ligands are upregulated. on activated monocytes and dendritic cells. PD-Li can bind to either PD-1 or B7-l. The fact that PD-1 binds to PD-LI and PD-L2 places PD-1 in a family of inhibitory receptors with CTLA4, While engagement of a costimulatory'receptor results in a costimulatorv signal in an inune celL eugagenent of an inhibitory receptor, e.g., CTLA4 or PD-I (for example by crosslinking or by aggregation), leads to the transmission of an inhibitory signal inan immune cell, resulting in downmiodulation of immune cell responses and/or in immune cellanergy. While transmission of an inhibitory signal leads to downnmodulation in immune cell responses (and. a resulting downmodulation in the overall immune response), the prevention of an inhibitory signal (eg. by usinga non-activating antibody against PD-1) in immune cells leads to upmodulation of immune cell responses (and a resulting upmodulation of an immune response). Despite the fact that PD- ligands suchas PD-.,are usually membrane-bound polypeptides expressed on professional immunological cells, it is known that such ligands can naturally be expressed in a soluble form (i.e. lacking a cellular membrane retention domain, such as ahydrophobic trasmembranedomain) and can be expressed by avariety of cells, such as cancer cells. For example, Frigola et al ) (2011) Cin. Cancer Res 1^7:1915 describes theassociation ofasoluble form of PD-LI with aggressiverenalcellcrinoma. Such soluble versions of PD-i ligand (eg- soluble PD-L1) provide new biomarkers associated with the detection of maladies and allow fbr a more non-invasive evaluation of cancer status due to easier detection of the biomarker in a bodily fluid as opposed to cancerous or pre-cancerous tissue, However, such protein isoforms are thought to be cleavage products of menbrane-bound PD-LI, as opposed to products of alternative splicing and transcript variants. Accordingly, there is a great need to identify additional PD-1 ligand (e.g, soluble PD-L)-based biomarkers associated with deleterious conditions in subjects, including the generation of diagnostic, prognostic, and therapeutic agens to effectively control such disorders in subjects.

Summary of the Invention The presem invention is based, at least in part, on the discovery that PD-LI isofonrs, particudarly thosc encoding soluble forns of PD-L, are overexpressed by specific cancers (e.g. head, neck, and lung cancers) and maintain the ability to transmit inhibitory signals to immune cells to thereby inhibit immune responses(eg T cell aciivation, proliferation, and cytotoxic function). Such PD-Li isoforns and the encoded. PD-L1 variants are useful as biomarkers for the identification,assessment, prevention, and/or treatment of such cancers. In one aspect, a method of determining whether a subject is afflicted with a head, neck, or lung cancer or at risk for developing a head,neck, orJung cancer is provided, wherein the method comprises: a) obtaining a biological sample from the subject; b) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in TableI or a fragment thereof in the subject sample; c) determining the copy number, level of expression, or level of activity of the one or more biormarkers in a control; and d) comparing the copy number, level of expression, or level ofactivity of said one or more biomarkers detected insteps b) and c); whereina significant increase in the copy number, level ofexpression, or level of activity of the one or more biotarkers in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more biomarkersm indicates that thesubjectis afflicted with the head, neck, or lung cancer or is at risk for developing the head, neck, or lung cancer. In another aspect, a method of determining whether a subject afflicted with a head, neck, or lung cancer or at risk for developing a head, neck or lunL cancer would benefit from modulating PD-I and/or PD-ti levels is provided, wherein the method comprises: a) obtaining a biological sample from the subject; b) determining the copynumber, level of expression,or level of activity of one or niore biomarkers listed in Table I or a fragment thereof in the subject sample; c) determining the copy number, levelof expression, or level of activity of the one or more biomarkers in a control; and d) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps b) ande;whereinasignificant increase in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more biomarkers indicates that the subject afflicted with the head, neck, or luing cancer or at risk for developing the head, neck, or lung cancer would benefit from modulating PD-LI and/or PD-LI levels. In stillanother aspect, a methodfor otoring th ression ofaheadneckor lung cancer in a subject is provided, wherein the method comprises: a) detecting in a subject sample at a. first point in time the copy number, level of expression, or level of activity of one or more biomarkers listed in'Table 1 ora fragment thereof; b) repeating step a) at a subsequent point in time; and c) comparing the copy number, level of expression, or level ofactivity of said one or more bionarkers detected in steps a) and b) to monitor the progression of the head, neck, or lung cancer, wherein the one or more biomarkers comprises soluble PD-L1, optionally wherein the soluble.PD-L a) comprises an amino acid sequence of SEQ ID NO:13 or 15 or an arnino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or b) comprises anucleic acidmolecule comprising a nucleic acid sequence encoding an amino acidsequenceof SEQ ID NO:13 or 15 oran aminoacid sequence that isat least 80% identical to SEQ IDNO:13 or 15. In one embodiment of any aspect of the present invention, an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample at a finst point in time relative to the copy number, level of expression, or level of activity of the one or more bionimarkers in the subject sample at a subsequent point in time indicates progression of the head, neck or lungcancer; or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level ofactivity of the one or more biomarkers in the subject sample at a first point in te relative to the copy number, level ofexpression, or level ofactivity ofthe one or more biomarkers in the subject sample at a subsequent point i time indicates a lack of significant progression of the head, neck, or lung cancer. In another embodiment the subject has undergone treatment to modulate PD-1 and/or PD-Li levels between the first point in time and the subsequent point in time.

In yetanother aspect, amethod for stratifying subjects afflicted with a head, neck, or lung cancer according to predicted clinical outcome of treatment with one or more modulators of PD-1 and/or PD-Li levels is providedxwhcrein the method comprises: a) determining the copy number, level of expression, or level of Ictivity of one or more biomarkers listed in Table I or a fragment thereof in a subject sample; b) determining the copy number, levelfxpression, or level of ac-iity of the one ormore biomarkers in a control sample; and c.) comparing the copy number, level of expression, or level ofactivity of said one or more biomarkers detected in steps a) and b); wherein a significant modulation in the copy number, level of expression or level of activity ofthe one oore biomarkers in the subject sample relativeto the normal copy number, level of expression, or level ofactivity of the one or more biomarkers in the control sample predicts the clnical outcome of the patient to treatment with one or more modulators of PD-I and/or PD-LI levels.ioneembodiment,thepredictedclinicaloutcomeis(a)celluar grh(b) cellular proliferation, or (c) survival time resulting from treatment with one or more 1.5 modulators of PD- and/or PD-.1 levels. In another embodiment, the one or more biomarkers comprises soluble PD-Lit optionally wherein the soluble PD-LI a) comprises an aminoacid sequence of SEQ ID NO:13 or 15 oran amino acid sequence that is at least 80% identical to SEQID NO:13 or 15; or b) comprises anucleicacid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ IDNO:13 or 15 or an aminoacidsequencethatisatleast80%identicaltoSEQIDNO:.In still another embodiment, an at least twenty percent increase or an at least twenty percent decrease between the copy number.level of expression, or level of activity of the one or more biomarkers in the subject sample compared to the control sample predicts that the subject has a poor clinical outcome; orwherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in the suibect sample compared to the control sample predicts that the subject has favorable clinical outcome. In yet another embodiment, the methodfurther comprises treating the subject with a therapeutic agent that specifically modulates the copy mimber,level of expression, or levelof activity of the oneor more biomarkers. In another embodiment, the method further comprises treating the snuject with one or more modulators of PD-i and/or PD-L1 levels. In another aspect, a method of determining the efficacy of a test compound for inhibiting a head, neck, or lung cancer in a subject is provided, wherein the method comprises: a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 1 or a fragmntthereofinafirstsampleobtainedfrom the subject and exposed to the test compound; b) determining the copy nuriber, level of expression, or level of activity of the one or more biomarkers ina second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and c) comparing the copy iniumber, level of expression, or level of activity of the one or more biomarkers in the first and second samples, wherein a significantly modulated copy number, level of expression, or level ofactivity of the bionarker, relative to the second sample, is an indication that the test compound is efficacious for inhibiting the head, neck, or lung cancer in the subject. In one embodiment, the one ormore biomarkers comprises soluble PD-., optionally wherein the soluble PD-i a) comprises an amino acidsequence of SEQ ID NO:13 or 15 or an amino acid sequence thaisat least 80% identical to SEQ ID NO:13 or 15; or b) comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least identical to SEQ ID NO:I In another embodiment, the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject. In stillanother aspect, a method of determining the efficacy of a therapy for inhibiting a head, neck, or hng cancer in a subject is provided, wherein the method comprise: a) determining the copynumber, level of expression, or level of activity of one or more blomarkers listed in Table I oia fragment thereof in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject; b) determining the copy number, level of expression, or level ofactivity of the one or more biomarkers in a second sample obtained from the subject following provision of the portion of the therapy; and c) comparing the copy number, level of expression, or level of activity of tie one or more biornarkers in the first and second samples, wherein a sigificantydecreasedcopy number, level of expression.or level of activity of the one or more biomarkers in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the head, neck,or lug cancer in the subject. In one embodiment, the one or more biomarkers comprises soluble PD-LI, optionally wherein thesoluble PD-LI a) comprises an amino acid sequece of SEQ ID NO:13 or 15 or anamino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or b) comprises a nucleie acidmolecule comprising a nucleic acid sequence encoding an aminoacidsequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO: 1 In yetanother aspect, a method for identifying a compound which inhibits a head, neck, or lung cancer is presented, wherein the method comprises: a) contacting one or more bioiarkers listed in Table I or a fragment thereof with a test compound; and b) determining the efFect of the test compound on the copy number, level of expression, or level of activity of the one or more biomarkers to thereby identify a compound which inhibits the head, neck, or lung cancer. Inone embodiment, the one ormore biomarkers comprises soluble PD-L, optionally wherein the soluble PD-L Ia) comprises an amino acid sequence of SEQ ID NO:13 or 15 oran amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or b) comprises a nucleic acid molecule comprising a nucleic acid sequence encoding anamino acid sequence of SEQ IDNO:13 or 15 or an amino acid sequence that's at least 80% identical to SEQID NO: 1. In another enbodiment, the one or more biomarkers is expressed on or in a cell (e.,cells isolated roman animalmodelofahead,neck,orlungcancer or cells froma subject afflicted with a head, neck, or lung cancer), In another aspect, a method fbr inhibiting a head, neck, or ung cancer is provided, wherein the method comprises contacting a cell within. agent thatmodulates the copy number, level of expression or level of activity of one or more biomarkers listed in Table I orafragment thereof to thereby inhibit the cancer. In one embodiment, the one or more biornarkers comprises soluble PD-L1, optionally wherin the soluble PD-L Ia) comprises an amino acid sequence of SEQ IDNO:13 or 15 oran aminoacid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or b) comprises a nucleic acidmolecule comprising a nucleic acid sequence encoding an aminoacidsequence of SEQ ID NO:13 or 15 oran amino acid sequence that is at least 80% identical to SEQ ID NO: L hi another embodiment, the copy number, level of expression, or level of activity of the one or more biomarkers is downmodulated. In still another embodiment, the step of contacting occurs in vivo, ex vivoorinvitro. In etanotherembodiment, themethod further comprises contacting the cell with an additional agent that inhibits the head, neck, or lung cancer. Instill another aspect, a methodfor treatrin a subject afflicted with head, neck, or lung cancer is provided, where themethod comprises adinistering an aent that downregulates the copy number, level of expression, or leVel of activity of one or more biomarkers listed in Table I or a fragment thereof such that the head, neck, or lung cancer

-.9- is treated.inoneembodimenttheoneormoreiomarkerscomprisessolublePD-L], optionally wherein the soluble PD-Li a) comprisesan amnoacid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or b)comprises anucleic acid molecule comprisinganmucleic acidsquence encoding anamino acid sequence of SEQ ID NO:3 or 15 oranaminoacid sequence that is at least 80% identical to SEQ ID NO:1. in another embodiment, the method further comprises administering one or more additional agents that treats the cancer. In still another embodiment, the agent is one or more modulators of PD-l levels and/or one or more modulators of PD-Li levels. In yetanotheraspect, a pharmaceutical composition is provided comprisin aT antisense polynucleotide that specifically binds to a polynucleotide of one or more biomarkers listed in Table I or a fragment thereof useful for treating a head, neck, or lung cancer in a pharmaceutically acceptable carrier. In one embodiment, the antisense polynuceotide frther comprisesan expression vector. In another embodiment, a method of using such pharmaceutical composition for treating a head, neck, or lung cancer in a subject is provided. In another aspect, a kit comprising anagent which selectively binds to one or more biomarkers listed in Table 1 ora fragment thereof and instructionsfor use. In one embodiment, the agent is selected from the groupconsisting of polynucleotides and antibodies. In still another aspect, a biochip is provided comprising a solid substrate, said substrate comprising one or more probes capable of detecting one or more biomrkers listed in Table I ora fragment thereof wherein each probe is attached to the substrate at a spatially definedaddress. In one embodiment, the probes are complementary to a genomic or transcribed polynucleotide associated with the one ormore biomarkers. Certain embodiments can apply to one more than one, or all aspects of the present invention. For example, in one embodiment of my aspect of the present invention, the one or more biomarkets comprises solible PD-LI, optionally wherein the soluble PD-L i a) comprises anamino acid sequence of SEQ ID NO:13 or 15 or an aminoacid sequence that isat least 80%identical to SEQ ID NO:3 or 15; or b) comprises a nucleic acid molecule comprising a nucleic acid sequence encodingan amino acidsequenceofSEQID :13or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:1. In another embodiment, the control is determined from a non-cancerous sample from the subject or member of the same species to which the subject belongs, In still another embodiment, the sample consists of or comprises body fluid, cells, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, scrum, buccal scrape, saliva, cerebrospinal fluid, urine, stooL mucus, or bone marrow., obtained from the subject, In yetanother embodiment, the body fluid is selected from group consisting of anmiotic fluid, aqueous humnr, bile, blood and blood plasma, cerebrspinal fluid, ceramen and earwax, cooper's fluid or pre-ejaculatory fluid, chyle, chyme, stool. female jaculate, interstitialfluidintracellular fluid, lymph, menses, breast milk, mucus, pleuralfluid, peritoneal fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, and vomit, in another embodiment, the copy number is assessed by microarray, quantitatie PCR (qPCR), highthroughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH), in still another embodiment, the expression level of the one or more bioarkers is assessed by detecting the presence in the samples ofa polyncleotide molecule encoding the biomarker or a portion of saidpolynucleotide molecule (eg7 amRNA, cDNA, or functional variants or fragments thereof and, optionally, wherein the step of detectingfurther comprises amplifying the polynucleotide molecule). In yet another embodiment the expression level of the one or morebiomarkers is assessed by annealing a nucleic acidprobe with the sample of the polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions, In some embodiments, the expression level of the biomarker is assessed by detecting the presence in the samples of a protein of the biomarker,a polypeptide, or protein fragment thereof comprising said protein, such as by using a reagent which specifically binds with said protein, polypeptide or protein fragment thereof (g, an antibody, an antibody derivative, andan antibody fragment). In still another embodiment, the activity level of the biomarker is assessed by determining the magnitude of modulation of the activity or expression level of downstream targets of the one or more biomarkers, In yet another embodiment, the agent or test compound iodulates PD-1, PD-L1, and or soluble PD-Ll levels (e.g, compound inhibits the expression and/or activity of soluble PD L1, optionally wherein the soluble PD-1.I a) comprises an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least80% identical to SEQ ID NO:13 or 15; or b) comprises anucleic acidmolecule comprising a nucleic acid sequence encoding an aminoacid sequence of SEQ ID NO:13 or 15 oran amino acid sequence that is at least 8W/;%identical to SEQ ID NO:.1 Examples of such agents includea.) an antibody against soluble PD-LI, optionally wherein the soluble-PDL Icomprises an aminoacid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; b) a small molecule inhibitor of soluble PD-I1, optionally wherein thesoluble PD-L Icomprises an amino acid sequence of SEQ ID NO:13 or 15 oran amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15: andc) an anti-PD-L inhibitor selected from the group consisting of a small molecule, antisensenucleic acid, interferingRNAshRNAsiRNA, aptamer, ribozyme, and dominant-negative protein binding partner, In some embodiments, the head or neck cancer is squamous cell carcinomas of the head and neck (SCCHN) or associated with human papillomavirus infection, In another embodiment, the lua cancer is sniall-cell lung carcinoma (SCLC) ornon-small-celllung carcinoma(NSCLC). Instill another embodiment, the subjectisamammal. In yet another embodiment, the mammal is a human.

BriefDescription of Fiaures Figures 1-2 show a schematic of a national landscape of head and neck cancers from The Cancer Genone Atlas (TCGA;Figure 1) proWct oran independent cohort of HPV- (left half) and HPV+ (right half) head and neck cancers (Figure 2). Figure 3 showsa representation of sites of HPV integration in the host genome in head and neck cancers analyzed as part of the TCGA project described in Figure 1. Figures 4 and 5 show sequencing reads for the CD274 (PD-L1) gene in a umor (CV-5433) with a detected HPV integrationinPD-LI.Figure5showsazoomedinviewof Figure 4 according to a log scale. Figure 6 shows a consolidation of the sequencing eaid data of Figures 4 and 5 into a schematic showing the HPV integration within the PDI I gene of tumor CV-5433 Figure 7 shows the predicted protein product following HPV integration in tumor CV-5433. Figure 8shows expression of each exon of fil-lienth, membrane-bound PD-Ll on a log scale from the CV-5433 sample and demonstrates a dramatic drop in exos following exon 4 which is the site of HPV integration,

Figures 9-10 show transcript variants of PD-L Iexpressed by head, neck,and lung cancers. Figures 11A-iIJ show expression of short PD-LI forms among variouscancers fror the TCGA (The Cancer Genome Atlas) database. Figures 12 shows expression of short PD-Li forms among various cancers from the Cancer Cell Line Encycspedlia database. Figures 13-14 show Western blot results 293T cells transfected withexpression vectors encoding the full-length or short PD-Li fbrms Figure 15 shows the protein expression of short PD-L Iforns from various cell lines. Figure 16 showsT cell viability in response to exposure toshort PD-Li forms. Figure 17-18 shows that the short form of PDL t can differentially kill proliferating T cells.

Detailed Description of the Invention The present invention is based, at least in part, on the novel discovery of gene profiles useful for distinguishinu among cancer subtypes (ctg., head, neck, and/or lung cancers) and for predicting the clinical outcome of such cancer subtypes to therapeutic regimens, particularly to modulators of PD-1 and./or PD-Li Thus, agents such asmiRNAs, miRNA analogues, small molecules, RNA interference,apamer,peptides, peptidomimetics, antibodies that specifically bind to one or more biomarkers of the invention(eg, bioniarkers listed in Table 1) and fragments thereof can be used to identify, diagnose, prognose, assess, prevent, and treat cancers (e.ghead, neck, and/or lung cancers) or other conditions that would benefit from modulating immune responses,

I. Definitions The articles "a"and"an"are used herein to refer to one or to more than one (ie., to at least one) of the granmnatical object of the article. By way ofexample,"an element" means one element or more than one element. The term "allogeneic" refers to deriving from, originating in, or being members of the same species, where the members aregenetically related or genetically unrelated but genetically similar. An"allogencic transplant" refers to transfer of cells or organs front a donor to a recipient, where the recipient is the same species as the donor, The term

"misnatched ailogeneic" refers to deriving from originatininor being members of the

same species having nonidentical major histocompatability complex (MHC) antigens (i.e., proteins) as typically determined by standard assays used in the art, such as serological or molecularanalysis of a defined number ofNMHC antigens. A "partial mismatch"refers to partial match of the MHC antigens tested between members, typically between a donor and recipient. For instance, a "half mismatch" refers to 50% oftheM-HC antigens tested. as showing different NIHC antigen type between two members. A"full"or"complete" mismatch refers to all MHC antigens tested as being different between two members These terms contrast with the term "xenogeneic," which refers to deriving from, originating in, or being members of different species, eg, human and rodent, human and swine, human and chimpanzee, etc. A "xenogeneic transplant" refers to transfer of cells or organsfrom a donor to a recipient where the recipient is a species different from that of the donor. The tert "syngeneic" refers to deriing from, originating in, or being members of the sane species that are genetically identical, particularly with respect to antigens or imnmunological reactions. These include identical twins having matching MHC types. Thus,a"syngencie transplant"refers to transfer of cells or orgas from a donor to a recipient who is genetically identical to the donor, The term "altered amount" of almarker or "altered level" of a marker refers to increased or decreased copy number of the marker and/or increased or decreased expression level of a particular marker gene or genes in a cancer sample, as compared to the expression level or copy number of the marker in acontrol sample. The term "altered amount" of a marker also includes an increased or decreased protein level of a marker in a sample, e.g a cancer sample, as compared to the protein level of themarker in a normal, control sample. The "amount" of a marker, eg. expression or copy number of a marker or minimal common region (MCR), or protein level of a marker, in a subject is"sigificantly" higher or lower than the normal amount ofa marker, if the amount of themarker is greater or less, respectively, than the normal level by an amount-greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount. Alternately, theamount of themarker in the subject canbeconsidered"significntly" higher or lower than thenormal amount if the amount is at least about two, and preferably at least about three, four, or fivetimes, higher or lower, respectively, than the normal amount of the marker. In some embodiments, theamount of the marker in the subject can be considered "significantly" higher or lower than the normal amount if thle amount is 10%15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% ormore, higher or lower, respectively, than the normal amount of the marker. The trn "altrcd level ofexpression" of a iarkr refers to an expression level or copy number of marker in a test sample e.g., a sample derived from a su bect suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is prefierably at leasttwice, and more preferably three, four, five or ten or more tines the expression level or copy number of the marker or chromosomal region in a control sample (e.g., sample from a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker or chromosomal region in several control samples, The altered level of expression is greater or less than the standard error of theassay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker In a control sample (e.g., sample from a healthy subject not having theassociated disease)and preferably, the average expression level or copy number of the marker in several control samples. The term "altered activity" of a marker refers to an activity of a marker which is increased or decreasedin a disease state, e.g., ina cancer sample, as compared to the activity of the marker in a normal, control sample. Altered activity of marker may be the result of, for example, altered expression of the marker, altered protein level of the marker, altered structure of themrker, or, e.g an altered interaction with other proteins involved in the same ordiffentpathway as the marker, or altered interaction with transcriptional activators or inhibitors. For example, the term "PD-1 ligand (e.g, soluble PD-L1) activity" includes the ability of a PD-1 ligand(e.g.soluble PD-LI) polypeptide to bind its natural receptor(s) (eg, PD- or 137-1), the ability to modulate immune cell costimulatory or inhibitory signals, and theability to modulate the immune response. With respect to PD-1, the term "activity" includes the ability of a PD- polypeptide to modulate an inhibitory signal inan activated immune cell, ea., by engaginga natural PD-I ligand (e.g, soluble PD-LI.) on an antigen presenting cell- PD- transmits an inhibitory signal to an immune cell in a manner similar to CTLA4. Ndulation of an inhibitory signal in an immune cell results in modulation of proliferation of, and/or cytokine secretion by, an immune ell. Thus, the term "PD-I activity" includes the ability of a PD-I polypeptide to binditsnatural ligand(s), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

The term "altered structure" of a marker refers to the presence ofmutations or alleic variants within the marker geneor maker protein mutationswhich affect expressionor activity of the marker, as compared to the normal or wild-type gene or protein- For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the marker.

The term "atered subcellular localization"of a marker refers to themislocalization of the marker within a cell relative to the normal localization within the cell eg, within a healthy and/or wild-type cell An indication of normal localization of the marker can be determined through an analysis of subcellular localization motifs known in the field that are harbored by marker polypeptides. Unless otherwise specified herein, the terms"antibody"and "antibodies"broadly encompass naturally-occurring forms of antibodies (e.g, IgG IgA, Ig, IgE) and recombinantantibodiessuch as single-chainantibodies, chimeric and humanizedantibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragntems and derivatives have at least anantigenic binding site. Antibody derivativesmay comprise a protein or chemical moiety cnijugated to anantibody, The term "antibod"as used herein also includes an "antigen-binding portion" ofan antibody (or simply "antibody portion"). The teri "antigen-binding portion", as used herein, refers to one or more fragments of anantibody that retainhtbiliytospecifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed wwithin the temi "antigen-binding portion" of an antibody include () a Fab fragment, a monovalent fragment consisting of the VL, V, CL and CHI domains; (i) a F(ab')2 fragment a bivalnttfragnient comprising txoFab fragments linked by a disulfide

bridge at the hinge region; (iii) a Fd framentconsistingoftheVHandCHdomains;(iv)a Fv figmentcosisti of the VL and V domains of a singlearm of anantibody, (v)a dAb fragment (Ward eia, (1989)awure 341:544- 5 46), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR)- Furthermore, although the two domains of the Fv fragment, VL and V1 are coded for by separate genes, they can be joined, using recombinant ethds, by a synthetic tinker that enables them to te made as a single protein chaiw i Which the VL and VH regions pair to form monovalent polypptides (known as single chain Fv (scFv); see e.g, Bird et al (198)Sciencc 242:423-426; and

Huston el al (1988) Proc. NatlAcad, Si. S USA 85:5879-5883; and Osbourn et at 1998, Nature Biotechnology 16 77 8), Such single chain antibodies are also intended to be encompassed within the term "antien-binding portion" of an antibody, Any V and VL sequences of specific scFv can be lined to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes.V H and VL can also be used in the generation of Fab , F or other immunoglobulinsusing eitherprotein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, specific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complemntarydomains of another chainand creating two antigen binding sites (see Hollier P, et al.(1993)Proc NatLA cadStci.S 906444-6448; Poljak, R. , etaL (1994) Striwture2:1121-1123)_ Still further, an antibody orantigen-binding portion thereof may be part of larger imnmoadhesion polypeptidesformedbycovalentornoncovalenrassociationofthe anibodyor antibodyprtionwithoneormoreotherproeinsor peptidesExamplesofsuch imnmunoadhesion. polypeptides include use of the streptavidin core region to make a tetrameric sFv polypeptide (Kipriyanov, S.M, et aL, (1995) Hunan.Antibodkes and lybridomas 6:93-101) and use of a cysteine residue, marker peptideand a C-terminal polyhistidine tag to make bivalentand biotinylated scFv polypeptides (Kipriyanov, SM., e al (1994) Moi mmuno31:1047-1058). Antibody portions, such as Fab and F(ab')2

fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively,ofwhoeatibodis,Morover, antbods antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques, as described herein. Antibodies may be polyclonal ormonoclonal; xenogeeic, allogeic, or syngenic; or modified forms thereof (e.g, humanized, chimeric, etc-). Antibodies may also befflly human. The terms "monoclonal antibodies"and "monoclonal antibody composition",as used herein, refer to a population ofantibody polypeptides that contain only one species of an antigen binding site apable of imunoreacting with a particular epitope of a1 antigen, whereas the term "polycional antibodies" and"pollonalantibodycomposition"refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with particular antigen. Amonoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts. The term "antisense"nucleicacidpolvpeptidecomprisesanucleotidesequence which is complementary to a "sense"nucleicacid encoding a protein; e'g, complementary to the coding strand of a double-stranded cDNA polypeptide, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid polypeptide can hydrogen bond to a sense nucleic acid polypeptide. The term "autologousrefers to deriving fromor originating in the same subject or patient. An autologouss transplant" refers to the harvestingand reinfusion or transplant of a subject's own cells or organs. Exclusive orsupplemental use of autologous cells can eliminate or reduce many adverse effects of administration of the cells back to the host, particular graft versus host reaction. The term"bochip" refers to a solid substrate comprising an attached probe or plurality of probes of the invention, wherein the probe(s) comprise at least 1, 27 3, 4, 5,6 7, 8, 9, 10, 11, 12t 13,14, 15, 16, 17, 18 19 20,25, 30, 35, 40, 45 50 55, 6065, 70t75,80., 85, 90. 95, 100. 150 200 or more probes. The probcsmay be capable of hybridizing toa target sequence under stringent hybridization conditions. The probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used,witheitheroverlapping probes orprobesto different sections of a particular target sequence. The probes may be capable ofhybridizing totargetsequencesassociated with a single disorder. The probes may be attached to the biochip in a wide variety of ways, as will be appreciated by those in the art The probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip. The solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment orassociation of the probes and isamenable toat least one detection method. Representative examples of substrates include glass and modified or functionalized glass, plastics Oncluding acrylics, polystyrene and copolyners of styrene and. other materials, polypropylene, polvethylene, polybutylene, polyurethanesTefonJetc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics. The substrates may allow optical detection without appreciably fluorescing. The substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics. The biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the probes mia be attached using fumetional groups on the probes either directly or indirectlyusingainker. Theprobes maybeattached-to the solid support byeitherthe 5' terminusterminus, or via an internal nucleotide. The probe may also be attached to the solid support non-covalently, For example, biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, probes may be synthesized on the surface using techniques such as photopolymerization and photolithography. The term "body fluid"refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g,amniotic fluid,aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-eaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleuralfluid, peritonealfluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit) The termins ancr or""umor"or"hperproliferative disorder" refer to the presence ofcells possessingcharacteristics typical of cancer-causing cells, such asuncontrolled proliferation, immorality, metstatic potential, rapid growth and proliferation rate, and certain characteristicmorphologicalfeauresCancer cells are often in the form of atumor, but such cells may exist alone within an imal, or may be a non-tumorigenic cancer cell, suchas a leukemia cell. Cancers include, butare not limited toB cell cancer, e.g, multiple myeloma, Waldenstro's macrolobulinemia,the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gaimopathy, and inunuocytic amyloidosis, mlanomas breast cancer, Wg cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, uinary bladder cancer, brain or centralnervous system cancer, peripheral nervoussystm cancer, esophageal cancer, cervical cancer, uterine or endometrial cancercancer of tie oral cavity or pharynx liver cancer, kidney cancer, testicular cancer, biliary tract cancer small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer,adrenal gland cancer, osteosarcoma, chondrosarcomcancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to themethods encompassed by the present invention include human sarcomas and carcinomas, eg fibrosarcoma, mvxosarcoma, hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcona, lymphanginendotheiliosarCOma, synOVioma, mesothelioma,Ewing's wmaor, leiomyosarcoma, rhabdonyosarcoma, colon carcinoma colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,inedullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinona, seminoma, enbryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testictdar cancer, lung carcinona, small cell long carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, meduloblastoma, craniopharyngiona, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menUngiomaM, elanoma, neuroblastoma. reinoblastona; leukemiast c.g..aute lymphocyc leukemia and acute melocyic leukemia (myeloblastic, promyelocytic, iyeloonocyticmonocttic anderythroleukemia); chronic leukemia (chronic nylocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemavea, lymphoma (odgkin's disease and non-Hodgkin's disease), multiple myeloim, Waldenstron's macroobulinemia, and heavy chain disease. Income embodiments, tie cancer whose phenotype is determined by the method of the invention is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynccologic cancers, renal cancer, laryngeal cancer, lung cancer,oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments. the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. [n still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma(e. scrous ovaan carcinoma),or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serious endometricid, mucinous, clear cell, brener, or undifferentiated, In some embodiments, the present invention isused in the treatment, diagnosis, and/or prognosis oflyniphoma or its subtypes, including, but not limited to,lymphocyte-rich classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, anaplastic large cell lymphoma, diffuse large B-cell lymphomas, MLL* pre B-cell ALL) based uponanalysis of markers described herein. The term "classifying" includes"to associate" or "to categorize"a sample with a disease state. In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both, In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to classify theunknown disease state of the sample- 1 certaininstances, cassifyinthe sample is akin to diagnosingthediseasestate of the sample. In certain other instances, classifying the sample isakin to differentiating ite disease state of the sample fromanther disease state. The term "coding region" refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, .whereasthe term "noncoding region" refers to regions of a nucleotide sequence that are not translated into aminoacids (e.g., 5 and 3untranslated regions). The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleicacid strands or between two regions of the same nucleic acid strand. It is known that anadenine residue ofafirstnucleicacidregion is capable offorming specific hydrogen bonds ("base pairing") with a residue ofa second nucleicacid region which is antiparallel to the first region if the residue is thymine or uracil Similarly, it is known that a cytosine residue of a firstnucleic acid strand is capable ofbasepairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the tvo regions are arranged inan antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion ad. the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at leastabout 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second. portion.

More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. The tern "control" refers to any reference standard suitable to provide a comparison to the expression products in tie test sample In1 oneembodiment, the control comprises obtaining a "control sample" from which expression product levels are detected and compared to the expression product levels from the test sample. Such acontrol sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample orprevious sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cclls/tissucs isolated from subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from thesame organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, ora primary cells/tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitablesource, including but not limited to housekeeping genes,an expression product level range fromnonnal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set ofpatients with a certain outcome (for example, survivalfor one, two, three, four years, etc.) or receiving a certain treatment. It will be understood by those of skill in the art that such control samples and reference standard expression product levelscan be used in combination as controls in the methods of the present invention. In oneembodiment, tiecontrolmay comprise normal or non-cancerous celltissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receding a certain treatment, or for a set of patientswith one outcome versus another outcome. In theformer cas specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mcan oraverage of the reference standard expression level In another preferredn ebodiment, the control may comprise normal cells, cells from patients treated with combminon chenotherapy and cells from patients having benign cancer, In another embodiment, the control mayalso comprise a measured value for example,average level of expressionof a particular gene in a population compared to the level of expression of a housekeeping gene in the same population, Such apopulation may comprisenormal subjects, cancer patients who have not undergoneany treatment (i.e., treatment naive), cancer patients undergoing therapy, or patients having benign cancer. In another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of twogenesin the test sample and comparing it to any suitable ratio of the same two genes In a reference standard; determining expression product levels of the two or more genes in the testsample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the testsample, and comparing to any suitable control In particularly preferred embodiments, the control comprises a control sample which is of thesame lineage and/or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the 1.5 basis for predictingoutcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome. and the expression product levels from the test sample are compared to the control expression productlevel as the basis for predicting outcome. As demonstrated bythe data below, the methods of the invention are not limited to use ofa specific cut-poinin comparing the level of expression product in the test sample to the control. As used herein, the term "costimulate"" with reference to activated immune cells includes the ability of a costimulatorymolecule to provide a second, non-activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector ftmetion. For example, a costimulatory signal can result in cytokine secretion, eg, ina. cell thathas receivedaTcell-receptor-mediatedsignal nmunecells that have received a cell-receptor mediated signal, egviaan activating receptor are referred to herein as "activated immune cells." The term "diagnosing cancer"i cludes the use of the methods, systems, and code of the present invention to determine the presence or absence ofa cancer or subtype thereof in an individual. The term also includes methods, systems, and code fir assessing the level of disease activity in an individual.

As used herein, the term "diagnostic marker" includes markers described heroin whichare useful in the diagnosis of cancer, e,g, over- or under-activity, emergence, expression, growth, remission, recurne resistance of tumors before, during or after therapy. The predictive functions of the marker maybe confirmed by, eg, (1) increased or decreased copy number(e by FISH, FISH plus SKY, single-molecule sequencing, e.g., as describedin the artat least at I Biotechnol., 86-289-301, or qPCR), overexpression or underexpression (e.g, by ISH, Northern Blot, or qPCR), increased or decreased protein level (e.g, by MC), or increased or decreased activity (determined by, for example, modulation of a pathway in which the marker is involved), e.g. in more than about 5%, 6%, 7%, 8%, 9%,10%1, I1 12%, 13%,14', 15%, 20;25%, or more of human cancers types or cancer samples; (2) its presence or absence in i biological sample, eg, a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinial fluid, rine,stool, or bone marrow, from a subject, eg., a human, afflicted within cancer (3) its presence or absence in clinical subset of subjects with cancer (e.g, those responding to a particular therapy or those developing resistance). Dianostic markers also include "surrogate markers," e.g, markets whichare indirect markers of cancer progression, Such diagnostic markers may be useful toidentify populations of subjects amenable to treatment with modulators of PD-I and/or PD-L. levels and to thereby treat such stratified patient populations. A molecule is "fixed" or "affixed" to a substrate if itis covalently ornon-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g, standard saline citrate, pH 7.4) without a substantialfraction of the molecule dissociating from the substrate. The term "gene expression data" or "geneexpression level" as used herein refers to information regardingthe relative orabsolutelevel of expression ofa gene or set of genes ina cell or group of cells. The level of expression of a gene may be determined based on the level of RNA, suchas mRNA, encoded by the gene, Alternatively,the level of expression may be determined based on the level of a polypeptide or fragment thereof encoded by the gne.Gene expression data may be acquired for an individual cell, or for a group of cells such asa tumor or biopsy sample. Gene expression data and gene expression levels can be stored on computerreadable media, e.g, the computer readable medium used in conijunction with a nicroarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.

The term "gene expression signature" or "signature"as used herein refers to a group of coordinately expressed, gnes. The genes making up this signature may be expressed in a specific cell ineage, stage of differentiation, or during a particular biological response. The genes can reflect biolooal aspects of the tumors in which theyare expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncoeric nechanismsresponsible for the cancer The term "head cancer" and"neck cancer" refer to cancers arising from head or neck region or tissue, respectively. in general, it isa group of cancers originating from the upper aerodigestive tract,, mcluding the lip, oral cavity, nasal cavity, paranasal sinuses, salivary glands, pharynx, and larynx, Cancers of the headand neck are further identified by the area in which they begin: cancers of the oral cavity, cancers of the salivary glands, cancer of the paranasal sinuses and nasal cavity, cancerscof the phary'nx and cancerscof the larynx, The termt"oal city" includes the lips, the pharynx, the tongue, the gurs gingivaa), the lining Inside te checks and lips buccall mucosa), the bottom (floor) of the mouth under the tongue, the bony top of the mouth (hard palate). the soft palate and the small area behind the wisdom eth (retromolar area), The salivary glands include the glands under the tongue lowerjaw (submandibularand sublingual), in front of the ears (parotid Land), as well as in other parts of the upper digestive tract-minorsalivary glands.Theterm"paanaasinuses"refersto the small hollow spaces in the bones of theheadsurrounding the nose. The term "nasal cavity" refers to the hollow space inside the nose. The term"pharnx reesto the hollow tube that starts behind the noseand leads to the esophagus andthe trachea. The pharynx has three parts: "nasopharynx" the upper part of the pharynx, behind the nose; "oropharynx," the middle part of the pharynx, which includes the soft palate, the base of the tongue and the tonsils; and "hypophlrynx " the lower part of the pharynx. Theterm larynx" is also known as the voicebox, and is the passageway formed by cartilage below the pharynx in the neck. ft contains the vocal cords and the epiglottis, Thus, the term "oral cancer encompasses all malignancies that originate in the oral tissues, in particular to cancers located in any part of the mouth, including the lips, gum tissue (gingival), tongue, cheek linmg buccall mucosa) and the soft or hard palate, andfloor of the mouth, or in the pharynx, the top part of the throat. Sometimes, squamous cancer cells are also found in the lymph nodes of the upper neck.

Most head and neck cancers begin in the squamous cells that line the structures found in the headand neck and are therefore termed squamous cell carcinomas (SCC-N.), Because of this, head and neck cancers are often referred to as squamous cell carcinomas. Some headand neck cancers begin in other types of cells. For example, cancers originating from glandular cells are called adenocarcinomas. With approximately 500,000 newcases annuallyquamous cell carcinomas oFthe head and neck, the vast majority oFwhich arise in the oral cavity, represent the sixth most common cancers in theworld. This disease results in nearlyabout 1.000 deaths each year in the United States alone. The five-year survival rate after diagnosis for HNSCC remains considerably low (approximately 50%). This poor prognosis of squamous cell carcinoma patients is likely due to the fact that most patients are diagnosed at advanced disease stages, and often fail to respond to available treatment options. As used herein a "squamous cell carcinoma" is a cancer arising, at least i part, frm a squamous cell population and/or containing, at least in part, a squamous cell population including, without limitation, cancers of the cervix; penis; head and neck, including, without limitation cancers of the oral cavity, salivaryglands paranasal sinuses and nasal cavity, pharynx and larynx; lung; esophageal; skin other than melanoma; vulva and bladder, Theterm"homologous" as uscd herein, refers to nucleotide sequence simiarity between two regionsof the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue positioninbothregionsisoccupiedbythe same nucleotide residue, then ihe reoins are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position ofeach region is occupied by the same residue. Ilomology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example,a region having the nucleotide sequence 5 ATTCCC-3'anda region having the nucleotide sequence 5'-TATGOC-3'share 50% homology, Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50% and preferably at least about 75%, at least about 90%, or at least about 95%ofthenuclotideresiduepositionsofeachofthe portions are occupied by thesame nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied bythe same nucleotideresidue, The term "host cell" is intended to refer to a cell iito which anucleic acid of the invention, such as a recombinant expression vector of the invention, has been introduced.

The terms "host cell" and "recombinant host cell"are usedinterchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progenyor potential progeny of such a cell, Because certain modifications mayoccur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The term "humanized antibody,"asused herein, is intended to include antibodies made bya non-human cell having variableand constant regions whichhave been altered to more closely resemble antibodies that would be made by a human cell, for example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in humangermiine immunoglobulin sequences. Humanized antibodies may include amino acid residues not encoded by human germiline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis inm tro or by somatic mutation in vivo), for example inthe CDRs. The term "humanized antibody", as used herein, alsoincludes antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as atmouse, have been grafted ontohuman framework sequences. As used herein, the term "immune cell" refers to cells that play a role in the imuie response. Immune cells are ofhematopoictic orinand include lymphocytes, such as B cells and T cellsnatural killer cells;myeloid cells, such as monocytes,macrophages cosinophils,mast cebasophis, and granulocytes. As used herein, the ten"inunune response" includes T cell mediated aid/or B cell mediated immune responses. Exemplary immune responses include T cell responses, eg, cytokine production and cellular cytotoxicity, In addition, the term immune response includesimmuneresponsesthat are indirectly effectedbyT cell activation, e.g, antibody production humorall responses) andactivation ofcytokine responsive cells, e.g, macrophages As used herein. the term "inhibit" includes the decrease, limitation, or blockage, of, forexampleapartilaraction,function,orinteractionFor example, cancer is "inhibited" if at least one symptom of the cancer, suchas hyperproliferativegrowth,is alleviated, terminated, slowed,or prevented. As used herein, cancer is also "inhibited" ifrecurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented. As used. herein, the term"inhibitory signal" refers to a signal transmitted via an inhibitory receptor (e,g, CTLA4 or PD-1) for a polypeptide on a immune cell. Such a signal antagonizes a signal via an activating receptor (e.g, via a TCR, CD3, BCR, or Fe polypeptide) and can result ine.g inhibition of second messenger mention; aninhibition of proliferation;an inhibition of effector function in the inunun cell, e.g, reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, thefailure of the immune cell to produce mediators, (such as cytokines (e.g, IL 2) and/or mediators of allergicresponses);or the development of anergy As used herein, the term "Interaction," wenreferring to an interaction between two molecules, refers to the physical contact (eg, binding) of the molecules with one another Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules. Alternatively, one or both molecules in the interaction may be prevented from binding their ligand, and thus be held inactive with respect to ligand binding activity(e.g binding its ligand and triggeingor inhibiting an inmnune response). To inhibit such an interaction results in the disruption of the activity ofone or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity. An "isolated antibody," as used hren, is intended torefer Wan antibody that is substantialLfree of other antibodies having different antigenic specificities, MTeover, an isolated antibody may be substantially free of other cellular material and/or chemicals, Asused herein, an "Isolated pron" refers to a protein that is substantially free of other proteis, cellular naterial, separation medim, and cultue medium when isolated. from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemicallysynthesized. The lanu subsumtialyfree of cellularrmaterial" includes preparations, in which compositions of the invention are separated fhom cellular components of the ellsfrom which they are isolated or recombinantly produced, In one embodiment, the laguagC "substantially free of cellular material'" includes preparations of having less than about 30%2, 10%, or 5 (by dry weight) of cellular material. When an antibody, polyept pepid fusion tin or fragment thereofag, abiologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, ie, culture medium represents less than about 20%., more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation, A "kit" Is anymanufacture (eg a package or container) comprising at least one reagent, ega probe, for specifically detecting or modulating the expression of a marker of de invention. The kii may be promoted, distributed, or sold as a unit for performing the methods of the present invention. As used herein, the term "hm, cancer" refers to the collection of cancers affecting~ lung tissue, Non-small cell lung cancer (NSCLC).represents approximately 87% of all lung cancers. The remaining 13% of all luing cancersare small cell lung cancers, though mixed-celllungcancersdooccur.Because smal eel lung cancer is rare and rapidly fatal, the opportunity for early detection is small There are rhree main types of NSCLC: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Adenocarcinoma is the most common form of lung cancer (30%-40% and reported.to beas high as 50%) and is the lungcancer most frequently found in both smokers and non-smokers. Squamous cell carcinoma accounts fbr 25-30% of all lug cancers and is generally foundin a proximal bronchus, Early sage NSLCtends to be localized, and if detected early it can ofen be treated by surgery with a favorable outcome and improved survival Other treatment options include radiation treatment, drug therapy, and a combination of these methods. NSCLC is staged by the size of the tumorand its presencein other tissues includinglymph nodes. In the occult stage, cancer cells are Found in sputum sarnples or lavage samples and no tumor is detectable in the lungs- In stage 0, only the innermostlining of thehugs exhibit cancer cells and the tumor has not grown through the lining. In stage IA, the cancer isconsideredinvasiveandhasgrown deep into the lung tissue but the tumor is less than cm across. In this stage, the tumor is not found in the bronchus or lymph nodes. In stage IB, the tumor is either larger than 3 cm across or has grown into the bronchus or pleura, but has not growninto the lymphnodes. In stage ILA, the tumor is more than 3 cm across and has grown into the lymph nodes. In stage IB, the tumor has either been found. in the lymph nodes and is greaterthan 3 cm across or grown into the bronchus or pleura; or the cancer is not in the lymph nodes but is found in the chest wall diaphragm, pleura, bronchus, or tissue that surrounds the heart, In stageIlA. cancer cells are found in the lymphnodes near the lung and bronchi ad. in those between the lungs but on the side of the chest where the tumor is located. In stage 11113, cancer cells are located on the opposite side of the chest from the tunor and in the neck. Other organs near the lungs may also have cancer cells and multiple tumors may be found in one lobe of the Iungs, In stage IV, tumors are found in more than one lobe of the same lung or both lungs and cancer cells are found in other parts of the body. Currentmethods of diagnosis forlun cancer include testing sputurn for cancerous cells, chest x-ray, fiber optic evaluation of airways, and low dose spiral computed tomography (CT). Sputum cytology has a very low sensitivity. Chest X-ray is also reaively insensitive, requiring lsions to be greater than I cm insize to be visible. Bronchoscopy requires that the tumor is visible insideairwaysacssibeto the bronchoscope, The most widely recognized diagnostic method is CT, but in common with X-ray, the use of CT involves ionizing radiation, which itself can cause cancer, CTalso has significant limitations: the scans requirea high level of technical skill to interpret and many of the observed abnormalitiesare not in fact lung cancer and substantial healthcare costs are incurred in following up CT findings. The most common incidental finding is a benign lung nodule. Lung nodules are relatively round lesions, orareas ofabnormal tissue, located within the lung and may vary in size. Lung nodules may be benign or cancerous, but most are benign, Ifa nodule is below 4mm the prevalence is only 5%if 4-8 mm the prevalenceis approximately 6%, and if above 20 mm theincidenceis aproxiately20%. F or small aindmedium-sized nodules, the patient is advised to undergo a repeat scan within three months toa year. For many large nodules, the patient receives a biopsy (which is invasiveand may lead to complications) even though most of these are benign. A "marker"orer includes a nucleic acid or polypeptide whose altered level of expression in a tissue or cell from its expression level in a control (e.g, normal or healthy tissue or cell) is associated with a disease state, such as a cancer or subtype thereof e.ghead,neckand/orlung cancers), A"marker nucleic acid" is a nucleic acid (eg, mRNA, eDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*,anti-miRNA. or a miRNA binding site, or a variant thereof and other classes of small RNAs known to a skilled artisan) encoded by or corresponding toa marker of the invention. Such marker aucleic acids include DNA (e.g, cDNA) comprising theentire or a partial sequence of any of the nucleic acid sequences set forth in Tablel and the Examples or the complement of such a sequence. The marker nucleic acidsalso include RNA comprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence, wherein all thyiidine residues are replaced with ridine residues, A "marker protein" includes a protein encoded byor corresponding to a marker of the invention. A marker protein comprises the entire or a partial sequence of any of the sequences set forth in Table I and the Examples or the Examples. The terms "protein" and "polypeptide" are used interchangeably. In some en bodiments, specific combinations of biornarkers are preferred. For example, a combination or subgroup of one or more of the biomarkers selected from the group shown in Table. As used herein,ihe term"modulate" includes up-regulation anddown-regulation, ag enhancing or inhibiting a response. The "normal" or "Control" level of expression of a marker is the level of expression of the marker in cells of subject, e.g., a human patient, not afflicted with a cancer. An "over-expression"or"significantlyhigher levelof expression" ofa marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and ispreferably at least twice, and more preferably 2.1, 2.,2.,2.5, 26 2.7,2.8, 2.9, 3, 35, 4, 4.5, 5, 5.5, 6, 65, 7,758, 85, 9, 9.5, 10, 10.5, 11, 12, 13 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the marker in a control sample (e.g samplefrom a healthy subject not having the marker associated disase) and preferably, the average expression level of the marker in several control samples. A "sigvnficatIy lOwerlevel ofexpression of marker refeTrs to an expressionlevel in a test sample that isat least twice, andmore preferbly 2.1, 2.2 2.3, 24, 25, 26, 27, 2,8, 29, 3, 3,5, 4, 4.5, 5.56,56. 7, 75, 8, 8,5, 9, 9.5, 10, 10,5, 11, 12 13, 14, 15, 16, 17 18,19, 20 times or more lower than the expression level of themarker in acontrolsample(eg, samplefrom ahealth'ysubject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples. The teri "pre-ralignant lesions" as described herein refers to a lesion that, while not cancerous, has potential for becoming cancerous. It also includes the term "pre malignant disorders" or "potentially malignant disorders." In particular this refers to a benign, morphologically and/or histologically altered issue that has a greater than normal risk of naignant transformation, and a disease or a patients habit that does not necessarily alter the clinical appearance of local issue but is associated with greater than normal risk of precancerous lesion or cancer development in that tissue (leukoplakia, erythroplakia, erytroleukoplakia lichen pLnus (ichenoid reaction) and any lesion or an area which histological examination showed atypia of cells or dysplasia.

The term "probe" refers to any molecule which is capableof selectivelybinding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker, Probes can be either synthesized b one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes Inlude, but are not limited toRNA, DNA, proteins, antibodies,and organic molecules. The term "progns includes a prediction of the probably course andoutcomeof cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individualFor example, the prognosis can be surgery, development of a clinical subtype of cancer (eg, head, neck, and/or lung cancers), development of one or more clinical factors, development ofintestinal cancer, or recovery froni the disease. The term "response to cancer therapy"or"outcome of cancer therapy" relates toany response of the hyperproliferative disorder (e.g cancer) to a cancer therapy, preferably to a change in tumor mass and/or volume after intiation ofneoadjuvant oradjuvant chemotherapy. Hyperproliferative disorder response may beassessed, for example for efficacy or in a neoadjuvant or adjuvant situation,where the size ofa tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation, Response may also be assessed by caliper measurementorpathological examiflation of theiurnor after biopsy or surcal resectin for solid cancers. Responses may be recorded in a quantitative fashion like percentage change intumor volume or in a qualitative fashion like "pathological complete response" (pCR) "clinical complete remission" (CR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (6PD) or other qualitative criteria. Assessment of hypcnroliferative disorder response may be done carly after the onset of neoadjuvant or adjuvant therapy, eg, after a few hours, days, weeks or preferably after a few inonts. A typical endpoint for response assessment is upon temination of neadjuvantchemothrapy or uipon surgical removal of residual tumor cells and/or the tumor bed. This is typically threemonthsaftrinitionofneadjuvanttherapyInsome embodiments, clinical efficacy of the therapeutic treatments described hereinnay be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured bv determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out fromthe end of therapy. The shorthand. for this formulas CBR:::CR+PR-SD over 6 months. In sone embodiments, the CBRfor a particular cancer therapeutic regimen is at least 25%, 30%, 35% 40, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%or more. Additional criteria for evaluating the response tocancer therapies are related to "survival," which includes all of the following: survival until mortality, also knownas overall survival (wherein said mortait may be either irrespective of cause or tumor related); recurrence free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include canticer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g, time of diagnosis or start of treatment) and end. point (e.g, death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to copy number. level of expression, level of activity, etc. of one or more biomarkers listed in Table I and the Examples or the Examples that were determined prior to administration of any cancer therapy. The outcome measurement may be pathologic response to therapy gien in the neoadIuvant setting. Alternatively, outcome measures, such as overall surivaland disease-free survival can be monitored overa period of time for subjects following cancer therapy for whom the measurement values are known. In certain enmbodinents, the same doses of cancer therapeutic agents are administered to each subject In related embodime-nts, the doses administered are standard doses known in the art for cancer thrIapeuticigents. The period of timefor which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker threshold values that correlate to outcome of a cancer therapy can be determined using methods such as those described inthe Exampiles section. Outcomes can also be measured in terms of a "hazard ratio" (the ratio of death rates for one patient group to another; Provides likelihood of deahat a certain time poit)t "overall survival" (OS), andor"progressionfree survival." In certain embodiments, the prognosis comprises likelihood of overall survival rate at 1 year, 2 years, 3 years, 4 years, or any other suitable time point. The significance associated with the prognosis of poor outcome in all aspects of the present invention is measured by techniques known in the art. For example, significance may be measuredwith calculation of odds ratio. In a further embodiment, the significance is measured byapercentage. Inone embodiment,a significant risk of poor outcome is measured as odds ratio of 08 or less or at leastabout 1.2, including by not limited to: 0- , 0.2,0.3,0.4, 0.5,0.6,0.7,0.8, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 4.0, 5.0, 10,0, 15,0 200, 25.0, 30.0 and 40.0. In a further embodiment, a significant increase or reductionin risk is at least about 20%, including but not limited to about'25% 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%, 70', 75%, 80%, 85% , 90%s,95% and 98%. In a further embodiment, a significant increase in risk is at least about 50%. Thus, the present invention further provides methods formaking a treatment decision for a cancer patient comprising carrying out the methods for prognosing a cancer patient according to the different aspects and embodiments of the present invention, and then weighing the results in light of other known clinical and pathological risk factors, in determining course of treatmentfor the cancer patient. For example, a cancer patient that is shown by the methods of the invention to have anincreased risk of poor outcome by combination chemotherapy treatment can be treated with more aggressive therapies, including but not limited to radiation therapy, peripheral blood stem cell transplant, bone marrow transplant, or novel or experimental therapies under clinical investigation. The term "resistance" refers toan acquired or natural resistance ofacancersample or mammal to a cancer therapy ( i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 2 or more, for example, 30%, 40%, 50%, 60%, 70%, 80%or more, to 2 fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, The reduction in response can be measured by comparing with thesame cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment. A typicalacquired resistance to chemotherapy is called "multidnig resistance." The multidrug resistance can bemediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when mammal is infected with a multi-dru-resistant microorganism or a combination of microorganisms. The determination of resistance toa therapeutic treatment is routinein theart and within the skill of an ordinarily skilled cliician, for example, can bemeasured by cell proliferative assays and cell death assays as described herein as "sensitizing," In some embodiments, the term "reverses resistance" means that the use of a second agent in combination with a primary cancer therapy (e,g, chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume ata level ofstatistical significance (eg p<0,05) when compared to tumor volume ofuntIrated tumorin thecircumstancewhere theprimary cancer therapy (e,g, chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tmior volume compared to tumor volmne ofuntreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically. The term "sample" used for detecting or determiningthepresenceorlevelofatleast one biomarker is typically whole blood, plasma, serum, saliva, urine, stool (eg, feces), tears, and any other bodily fluid (e.g, as described above inder the defition of "body fluids"), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgical resection tissue. In certain instances, the method of the present invention further cnmprises obtanimg the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample. The term "sensitize" means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancerwith acancerthera( chenmotherapeutic irradiation therapy. In some embodiments, normal cells are not affected to an extent that causes the normal cells to be tinduly injured by the cancer therapy (e.g., chemotherapyorradiationtherapy), An increased sensitivity ora reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, includin, but not limited to, cell proliferative assays (Tanigawa N,Kern D11, Kikasa Y,Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran EN., Cancer Res 1984; 94: 161-173; WeisendhalLI, LippmanM E Cancer Treat Rep 1985; 69: 615-632; Weisenthal L N, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: HarwoodAcademic Publishers, 1993: 415-432; Weisenthal L M, Contrib (ynecol Obstet 1994; 19: 82-90), The sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 61n1nth for human and 4-6 weeks formouse. A composition or a method sensitizes response to a theraleutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%,170%, 80%, or

3 35- more, to 2-fold, 3-fold, 4-fId, 5-fold, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatments routine in the artand within the skill ofan ordinarily skilled clinician- It is to be understood that any method described hereinfor enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise Cancerous cells (eg., resistant cells.) to the cancer therapy. The term "synerinstic effect"refers to the combined effect of two or more anticancer agents or chemotherapy drugs can be greater than the sum of the separateeffects of the anticancer agents or chemotherapy drugs alone The term "subject" refers to any healthyanimlmammal or human, orany animal, mammal or human afflicted with a condition of interest (e.gcancer). The term "subject" is interchangeable with "patient" The language "substantially free of chemical precursors or otherchemicals" includes preparations ofantibody, polypeptide, peptide orfusion proteinin which the protein is separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. I one embodiment, the language "substantiallyfree of chemical precursors or other chemicals" includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors ornon antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibodyi polypeptide, peptide or fusion protein chenicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and. most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals. The term "substantially pure cell population" refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about50%, preferably at least about 75-80, more preferably at least about 85-90%, and most preferably at leastabout 95% of tie cells making up the total ceil population. Thus, a "substantially pure cell population" refers to a population of cells thatcontain fewer than about 50%, preferably fewer than about2-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.

3 6-

As used herein, the term"survival" includes all of the following: survival until mortality, also knownas overall survival (wxherin said mortality may be either irrespective of cause or tumor related); "recurrence-ftee survival" (whihln the term recurrence shall include both localized and distant recrrence); metastasis free ura;disease free survival (wherein the term disease shall include cancer and diseassociated therewith), The length of saidsurvival rnv be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g- death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recturrence. A "transcribed pokynucleotide" or "nucleozide transcript" is a polymleotide (e,.g, an mRNA, hnRNA, cDNA, mature miRNA, premiRNA, pri-iRNA, miRNA*, anti miRNA, or a miRNA bindmingsite, or variant thereof or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of theinventionand normal post-transcriptional processing (C.g, splicing), if any, of the RNA transcript, and reverse transcription ofthe RNA transcript. As used herein, the term "vector" refers toa nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which referstoacircular doublestranded DNA loop into which additional DNA segments may be

ligated, Another iype of vector is a vieal vector,wherein additional DNA segments maybe ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.gbacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g,noc-episomal mammals vectors) areiintegrated into the genome of a host cell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred tn herein asrecombinantt expression vectors" or simply "expression vectors-, in general, expressionvectors of utility in recombinant DNA techniques are often in the form of plasmids. In the presentspecification, "plasmid"and "vector" may be used interchageably as the plasmid is the most coninonly used form of vector. However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.greplication defective reroviruses,adenoviruses and adeno-associated viruses), which serve equivalent functions. An "underexpression" or "significantly lower level of expression or copy number" of a marker refers to an expression level or copy number in a test sample that is greater than the standard error of theassay employed toassess expression or copy number, but is preferably at least twice, and. more preferably three, four, five or ten or more times less than the expression level or copy number of the marker in a control sample (e.g.., sample from a healthy subject not afflicted with cancer) and preferably, theaverage expression level or copy number of the marker in several control samples. As used. herein, the ter"unresponsiveness' includes refractivity of immune cells to stimulation, e.g. stimulation via an activating receptor or a cytokine U1nresponsiveness can occur, e,g., because of exposure to immunosuppressants or exposure to high doses of antigen. As used heriil thc termn"anergv" or "tolerance" inciudcs refractivityvto activating receptor-mediated stimulation, Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergyinT cells (as opposed to unresponsiveness) is characterized by lack of cytokine productione.g, IL-2. T cell energy occurs when T cells are exposed toantigen andreceive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal.), Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatorypolypeptide) results in failure to produce cytokines and, thus, failure to proliferate. Anergic T cells can however, proliferate if cultured with cytokines (etg, IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate IL-2 gene transcriptioninduced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a multimer of the AP. sequence that can be found within the enhancer (Kang ea ed(1992) Science -257:1134).

There is a known and definite correspondence between the anino acid sequence of a particular protein and the nucleotide sequences that can code for the proteinas defmed by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

Alanie (Ala, A) GCA, GCC, CG, GOT

Arginins A naagne (rg (A R,R) ) AG, AAC, ACCGIG AA GG G

Aspartic acid (Asp, 1)G GAT

GlA.umc aci (Glu E) GAA, GAGS Glut amne (Gin, Q) CAA, CAG

GlIne (Gl, G) GGA, GGC, G0GG0, GGT

istidine (His, H) A, CAT

le,I) ATA, ATC, ATT

Leucine (eu, L)1CA, CTC,' CTG,CTTA TG

(Lys, K) AAA, AAG Methionine (Met, M) ATG

Phenyla-lanine (Phe, F) TTC, TT

Prolirle (Pro, A) OCA, CCC, CCG, COT Serin (Ser', A) A c TA TC TG T

hreni.e (Thr, T) AA, A , ACG, ACT

TrptophaT (Trp, W) TGG

Tr(Tyr, Y) TAC, TAT

Valine(Val, 'V') GA TGG T rination sinal (end) FAA, TAG, TGA

An important ad. well known feature of the genetic code isits redundancy, whereby, for most of the amino acids used to make proteins, more than one coding 2$ nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the sameamno acid sequence in all organisms (although certain organisms may translate sonic sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found ina givennucleotide sequence. Such methylations do notaffect the coding relationship between the trinucleotide codon and the corresponding amino acid. i view of theforegoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the invention (or any portion thereof) can be used to derive the fusion protein or polypeptideamino acid sequence, using the genetic code totranslate the DNA or RNA into an amino acid sequence. Likewise, for a fusion protein or polypeptide amino acidsequence, corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from thegenetic code (which, because of its redundancy, willproduce multiple nucleic acid sequences forany given aminoacid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of afusion protein or polypeptide amino acid sequence hereinshould be considered to also include description and/or disclosure of all possible nucleotide sequences thIt can encode the amino acidsequence. Finally, nucleic acid andamino acid sequence information for the lociand biomarkers of the present invention (e.g, biomarkers listed in Table 1 and the Examples) are well known in the artand readily available onpublicly available databases, such as the National (enter for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below, The nucleic acid and aminoacid sequences of a representative human PD-I biomarker is available to the public at the GenBank database under NM005018.2,and NP 005009,2 (see also Ishida et al. (1992) EM )HOJ.1 1:3887; Shinohara e al. (1994) enoWmic-s 37 04;. and U.S Patent 5,698,520). Nucleic acid and polypeptidesequences of PD-I orthologs in organisms other than humans are well known and include, for example, monkey PD-1 (NM 001114358.1 andNP 001107830.1), mouse PD-1 (NM 0087998,2 and NP 032824I), rat PD-1 (NM_001106927.1 and NP 001100397,1), chicken PD-I (XM_422723-3and XP422723.2), cow PD- (NM 001083506.1 and NP001076975.1), and dog PD-l (XM5433383 and XP_543338&3) At least five transcript (i.e., splice) variantsencoding different human PD-Li isoforis exist and are described herein. PD-Ll proteins generally comprise a signal sequence, an IgV domain, and an gC domain. The sequence of human PD-L transcript variant I is the canonical sequence, all positional information described withrespect to the remaining isoforns are determined from this sequence, and the sequences are available to the public atthe GenBankdatabase underNM 014143.3andNP 054862.1 Inthis isoform, the signal sequence is shown from about amino acid I to aboutamino acid 18, the igV domain is shown from about amino acid 19 to about amino acid 134, the IgC domain is shown from about amino acid 135 to about amino acid 227, the transmembrane domain is shown from about amino acids 239 to about amino acid 259, and the cytoplasmic domain is shown from about amino acid 260 toabout amino acid 290. The sequences of human PD-L Itranscript vacant 2 can befound under NM 0012677061 andNP 001254635.1 and the encoded protein lacks an alternate in frame excon in the 5' coding region compared to variant (i.e., missing aminoacid residues 17-130) so as to result ina shorter protein The sequences of human PD-Li transcript variant 3 is provided herein and encodes a naturally occurringB7-4 soluble polypeptide, .e, having a short hydrophilic domain and no transmembrane domain. In this isoform, the signal sequence is shown fromabout amino acid I to aboutamino acid 18, the IgV domain isshown from aboutaminoacid 19 toabout amino acid 134, the 1gC domain of SEQ ID NO:2 is shown from aboutamrino acid 135 to about aminoacid27, and thehydrophilic tailis shown from about amino acid 228 to about amino acid 245. in addition, another soluble PD-L Iisoform exists having the amint acid sequence shown herein, This fourth PD-Li isoform differes from that of the first PD-LI isofbra in that there is a K to Dsubstitution ataminoacid position 178 andamino acid residues 179 290are deleted Moreover, another soluble PD-L Iisoform exists having the amino acid sequence of residues 1-227 encoded by transcript variant I and thereby unly comprising a signal sequence, the IgV domain, and the IgC domain In some embodiments, the soluble PD-L isoforis of the present invention do not contain the signal sequence as such a sequence is usually cleaved prior to secretion of the polypeptide from the cell. In other embodiments, the soluble PD-Ll isoforms of the presention inventionconsist of the IgV domain and the IgC domain (ie the extracellular portion of the full-length, membrane-bound PD-L1) and can further comprise heterologous sequences, such as Fe domains, protein tags, conjugated therapeutics, and the like. Such soluble PD-Li isoforms can be generated by alternative splicnin a number of ways well known to theskilled artisan involving the elimination of exons 4, 5, and 6 of full-length, membrane-bound PD-Li cDNA. Nucleic acid and polypeptide sequences of PD-L I orthologs in organisms other than humans are well known and include, for example, monkey PD-Li (NM_001083889.1 and

NP001077358,), chimpanzee PD-LI (NM_001140705,2 and XP-001i40705,i) mouse PD-LI (NM(021893.3 and NP068693.1), rat PD-LI (NM_001191954.1 and NP 001178883,1), chicken PD-LI (NM4248113 and XP_4248113), cow PD-LI (NM 001163412. 1and NP 0011568841), and dog PD-L I (XM_541302.3and 5 XP541302 3 Antibodies for the detection of PD-L Iand methods for making them are known in theart,

Table 1 Nucleic Acid and Amino Acid Sequences of the Present Invention

SEQ ID NO 1 Human PD- I cDNA Sequence

---------------- a------Cc -C: C tg oagtgg. aCgaagg g<gQaoaag aCq c C.Ctt oaC.t gagC.tt c C.aa c ao Catg bS 1I g aga C 6. cog t"j aaa C:ts ga C:C Nocagga... g .t ag :2" ceCasC:ja a C:C:2c .g ga agts eeggac acc&qacett ctecuccge tet<agactecccagaoag

301 gga Cocagg t t crgagegeg gcgg. gag -ataa ga getg.ga

a2g g a-agag -agggagc -C-c 'ag' ..... N<N z . C g C C C$ZuQ.- a gz<g C C4 : Q.- C.'C: t-- . Z.: C V 4q agcagt geggttc qatgt qttgtgtg tggga ggcc:t getgggCage

2t 7 g tecog 72 C tggg g ggggYet g a cge tcg ag agt ceeagtgY'a grrcac Y cagccc . atCtg tt..c. ctgag t gg.t: ageg agg

c-1 gag t gtettgCc j . ca torga : cC:tt , ~ aC ,0 Ca3R C~ C 4.~~~-'..

SEQ ID NO 2 Human PD-1 Amino Acid Sequence

3N0 61 es1vinwyrm spz gtdkla a2pedrsqpg gd p m -121 y aa iap kagi.kesi'a elr era vpapf rpagiti vgg gllgs

40 law icpr t ga-t gp e p v q

SEO ID NQ; 3 M4ouse PD-1.cDNA Sequence

C. attgg t c gacagac ttC.C:Catta aacttC.getg Cgcae gaegC C 2.aggg.gge tte.agaggccaatggg tggaggt CZaac tCaC eccag-o qg21 tgca Cag rgtcagaggg agcaate cetectgaetgocatg 1-t t- ta., -NatC:, C.t - c:C

2 41 gCtct ceacgecgagc ccgt augtta aaCag 30 caagge atgatta cagaat c ttgaca ac ggCg atg a C.ggg atk

-g 3 aact ::gtg gggat ag Cg tgg a4 cotttpgacg

a.,aaaCCagaggtta ag g e a t

SF0,E ID NO: 4 Mouse PD-1 Xrino Acid Scquinu.c 10 Q cdl -- "'.-I -a7 fIOXX1V0"'-cl `, I

0; ------- f :z 3nf-ar -kdl c----- 4 - '- v y-A -

, 15 SE)IJD.NO5, HummP D-LIVariant. IDN\%xouuice

121 aaccaccag a . cc, tzgag a C t --- ' <-

20 gK1 .'aC-k t-C---- a)t' ,a -:X' . -JCcy -I g-4 -

361~ N' qv'atc a a -g --- aat :C.at 3aaC .

,

4825cagaga c-30a-?: c t -caa~ -- ,g ,cc :- -an, a cC:a :

25 51 acawaa ocagagaa g a g g ct tc a C .. a C ac a - t q,-a t <-> a C

SE0 ID NOY 6 IHuman PEYL1 Isotonn I Amino Auid Sequence

nei fr pee .C pp- e~ot

SEO ID NO: 7 Human P.D- V1\ariant 2 eDNA S -quenct.

C.-,4 t <0qaC.'4---N---~4 a-"-- at-- gaa a aag - ---

45t g' a-------. oa a a c aCa 8 C. t a 8 -t 1,o a 1 ,-. t-1 t g

45 30 -acaotag aaaccta -CZl3O $Yatt( q c <t a att cc t<: tO~cto

41.ct tca SVc ,&011t040a0.a a -~a' 7--0 aaaQ0400: a ac

SEQ ID NO: 8 Iihana PD~LI Isofonm 2 Amio A cid Sequence S a ta'x.N',m -. ar ae Ncer sr4n'e nylAang.C 5 " '21 p 'ert rN'i N gailiel va i """riK" r"mav'"' e "eni"os tiee-t

SE ID NO: 9 1lunan PD I Isoform 3 cDNA Sequence gettccgg geeg aC aggcgtt CtgtecgCat geagggcatn co~agaaag SS 10 arg agg ta oett gt grC eto? ate etc atg acm tac-- gg ' at tt g r 10 Met Arg lie Phe Ala Val Pne lie Phe Met Thr Tyr Trp His Lea Leua

1.5 a ac g-a on: act gtm acg gt cc aag gae m'a tat gcga ta gag ta t 154 Amn Ala Phe Thr, -,,a Ih a Pro Lys Asp iLeu Tyr Val Val Giu Tyr,

220 25'30 ggt agc aar atg aca att gaa tgC aaa ttC CCA gta gaa 88a caa ,tra 2012 Gly Ser Asn Men Thy .ile Giu Cys Lys IPhe Pro Val Giu Lys GinLu

gac ctq act aCa cta atnt a t c at taa gaa atg gag gas aac :aac att 250 Ap Ala Ala Leu lie Val Tyr Trp Giu Met Giu Asp Lys Asn .le sa5 o

att Caa att gtg cat gga gag gaa gaecOng aag gtt Cag cat age agc 2r8 "le Gin he Val i.s Gy Giu Giu Asp Leu Lys Val Gin His Ser %Nr 6 VS 7,0 7 a'8

tac aga cag agg ge2 eg! cog ttg aag gac eag tC tec cog gga aat 3lE. Tyr Arg Gin Arg Ala Arg Leu Leu Lys Asp Gin Leu Ser Leu Gly Am

got -ca aot cag are sea gat gog aaa meg cag gat ges ggg gog tac 394 Ala Ala Leu Gin lie Thr Asp -,7ai Lys L eul- Gin , Asp Al Ia Gly -,,al- T'yr

cac vgC atg ate ago tat ggt act aCC gac tac aag Cga act act gtg 442. Arg Cys Wt .ie Ser Tyr Gly Gly Ala Asp Tyr L<ys Arg lie Thr Val 1W 120C2M

aaa gte aat gee Cca tao aac aaa atec iC a ;tiga antt ttg gte gtg 490k Lys Val Asn Al].a Pro, Tyr As-' Ly Pie An Gn Arg Ile Leu Va Va 13 l) 130 14 C

gat Cca gec ac Ocet gaa cat ia a, :g ac a tt g ge-t gag ggeca a"b Asp Pr o VlThr Ser G0u His Giua Leu Thr Cys Gin Ala Giu Giy Tyr 50 C Cc aag gee- gall gte are tgg aca age agt g-ac cat caa gt ctg agqt s Pro Lys Ala Gin Val lie Trp Thr Ser Set Asp His Gin Val Lau Ser

55 gQ eang acc :2o acc a .Cc ant too ang aga gag gag aag ctt etc act 13 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Gin Lys Leu Phe Amsn 18N 18 190

gtg acc ago aca ctg aga a'c aac a a act aa gag at, Wt tac U 2 Val Thir Ser Thr Leu Arg lie Asa Thr T r Thr Amn Glu le Phe Tyr tge acot tt- agg aga tta gat Ccet gag gaa aac ca- ac~a geaaa -- a 73 Thch'rgAgLu s ry,r Gi-,u A-nEi z:u hr A1 l e tct oca ggt a at a- Ctg a a gt EC. at aa a a atat ra aca 77 " lz PC Gv As Tle La u Asn Va.r Lysl> Cs Lu3. r as ~Aac taq:3:333533 iRtaagcattcat c taa

SEQ IDNO 10 urroan PD-b I Isotorm 3 Xmio Acid SSernT

Asv P.g Ae a n na4 'a ne 7 Ln e-, 7y) ,Iv al i1uLaSeu

Gy Ser e h nIa i y Lycs ValGi G Ir Lys7 Gn Leu, 44

Asp Leu, Ala a L7eiu Ilj VTr Trp T el s LysAn e

1l ci 1 h 1 val Gly Gu lu p Leu 1y Val Gln His 1 e sr 65 ^. 75 8-0 Tyr A rg Gn Arg eu, Alkrg Leu1 Lys Asp !., - Gn eu Ser Leu Gl AI

Ala la LuGn Il hr Aspn valIy Leu Gn As p Ala GIL'y Val Ty

Ar, -e',e r'v Tyr 'I! Iy Ala Ap Tyr y Ag l hrV

i val Se r Gu -Lu y l Ia l -y.yr

Ala Giu Va -er A's is na

Gl3 y y T TI, r Tu An L A-- GI u L Lys Lu Ph e A-N

Vl S hrL Argl A-n T Tr "Th A:n Giu lhezll T N2'- '^ 45

-45.-

)va.line kPro wiy eAsa LC Leu Aesn va.ser ne tys u1 cys Leu n

Lu Pro Ser The Prcr

15 SEQ ID NO;, .11 Human PD-LI Isoform 4 Am-i'no.Acid Sequence

SEQ ID NO: 1 2 Human PD-L1 Isoform 5 cDNA Sequence

-A-- GT neAGGu A-GA GGCiGTb;Asa-aiwuA

SEQ IDNQ: 13 mu PDFL hI fo 5 Amino Acid Segence (SolubleI

R' RIT A V-L T NC- V E 4-'

40 iC'cacAATGPTA KE ID 1 NO 4 GcT XGA.APDEGG Human VcA LScATTraAG 5 3?,a isoom 6 .DN A SeQwne ATA.- A CK s- -TIT A'TAT y ir A - T Vc K NTA PCT.'TAT- -I.....T A 7 40 '.-.u .--- oilia.." -r' K'------- 222:5t C'W~tt..45ksna tatin>.\....4.\...4--'u 7n .'.7"

"............-..-.'...-..'A-.4 .. A 7.e 4 a A 's-~--,u-.,soC-- ' .. - -.- e.

45' . N ., k Y : 7 AD N3 ..... ,

50 S SEQ .,., IA CIDGNO: 15 E 2.k T -C A G-A Human I sT G A T, TIvT Isoform A C AT AVT" I'C PD- -A TA C C 6 - A Amino AcidSeuence (Soble 2G S , G CA T, A LA A A C TS2- T s- G G

SEQ ID NO: 16 Mouse PD-LI eDNA SAunce

5 46 -

1 atgggare togegg a t ga ttec qcectqc acgetc gasat

11agattccctg tagaaCcggga gCtgqaCCt'g ettecgttag tggtgrga Ctg ggaaaaggaa S8 gaagaca gatt Ccagt tg gg ca gga gagggc tCag t a ccaaac 24 I-Cg -agceceget ecaaagga c %a"sxctt kg ega sggaaz2jtg egcca

3 cg ac 7taca agegC"aatccg"aateatcca accc'aaa'at cscgg 2attt g aC Cca Ca t agt-I .aattC va t a ggC-- g-ggtt-:'za"C estgaagetgaggtaega acggeccaee ggagaggatt z C aC: g aag g g t agt Cca gcg...ag ggtag .0l nac 8g cgaantg ag tenrer c ta cnrget t eg pggjac 'ag ca -ga a.Ia Ccacac 661 : geggagergCa teci'.C'.cg cjtge--fctC% a a Ccj;atect c r :a a'gaca .I -g gaeteanctg g g2 t g -g t tgagg Ctc 731gaaacag - gjag -g a gaga g a:ag2ge ctaaacC:;IageCa : 2 ;1a a aaag aaata a t-"a C-.a as t 'gagga-".aa aa

SEO ID NO: 17 Mouse PD-L I Amno Acid Seouenrce f rragCl -'I t aCCh IaIra ft eap kdi vv ey,-g:nyrmev -rpveed l.- - I Ia IVvyweke 61 degivI-q ag edikpqhcn ffrgrzasipkd: qllIkganaal I-Jt-dyvqdag vycciisy g g m adaitiak a a pyri)g isepee elgagy t eaev wJ. 8 we8tnd kgp nkrav 24v vlgilf ivt-v if rk vrmidr c e dts nrndeqe5

I11 Agents and Compositions Novel agents and compositions of the prent invention are provided herein and can be used for the diagnosis, prognosis, prevenion, and treatment of cancer (e.g., head, neck, and/or lung canicers) and cancer subtypes thereof Such agents and compositions can detect and/or modulate, eg, up- or down-regulate, expression and/or activity of gene products or fragments thereof encoded by biomarkers of the invention, including the biomarkers listed in Tal andteExamples.Ixempragents include antibodies, smallmolecules peptides, pelptidominetics, natural ignds, and. derivatives of natural lig.nds, that can either bind and/or activate or inhibit procin biomarkers of the invention, including the biomarkers listed in Table I and the Examples, orfragments thereof; RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expressionand/or activity of the biomarkers of the invention, including the bioiarkers listed in Table 1 and the Examples, or fragments thereof In one embodiment, isolated nucleic acid molecules that specifically hybridize with or encode one or more biomarkers listedin Table 1 and the Examples or biologically active portions thereof are presented. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (i.e, cDNA or genonic DNA) and RNAmolecules (i.e., mRNA)andanalogsof the DNAorRNAgenertedusingnueotidenlogs Thenucleic acid molecule can be single-stranded or double-stranded. but preferably is double-stranded DNA, An "isolated" nucleic acid iolecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (ie sequences located atthe 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecules corresponding to the one or more biomarkers listed in Table I and the Examples can contain less than about 5 kb, 4kb, 3kb 2kb, I kb, 0,5 kb or 0,1 kb of nucleotide sequences which naturally flank the nucleic acidmolecule in gnomic DNA of the cellfrom which the nucleic acid is derived (L e. a head and neck or lung cancer cell). Moreover, an "isolated"nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized A nucleic acid molecule of the present inventioneg. anucleic acidmolecule having the nucleotide sequence of one or more biomarkers listed in Table I and the Examples or a nucleotide sequence which is at least about SO%, preferably at leastabout 60', more preferably at least about 70%, yet more preferably at leastabout 8%, still more preferably at least about 90, and most preferably at least about 95% or more (e.g, about 98.) homoloaous to the nucleotid sequence of onie or more biomarkers listed in Table 1 and the Examples ora portion thereof(he, 100, 200, 300, 400, 450, 500, or more nucleotides), can be isolated using standard molecular biology techniques and the sequence information provided herein, For example, a human cDNA can be isolated from a human cell line (from Stratagene La Jolla, CA, or ClontechPalo.Alto, CA) using all or portion of the nucleic acid molecule.or frament thereof, asa hybridization probe and standard hybridization techniques ( e,as described in Sambrook, I, Fritsh, E. F., and Maniatis, T. Molecular Coig:A Laboratorv alnual 2nd, ed Cold!Spring HarborLaboratorg Cold. Spring Harbor LaboratoryPress, Cold Spring Harbor NY, 1989), Moreover, a nucleic acid molecule encomnpassing all or aportion of the nuleotide sequence of one or more biomarkers listed in Table I and the Examples or a nucleotide sequence which is at least about 50%, preferably at leastabout 60%, morepreferablv at leastabout 70%, vet more preferably at least about 80%, still more preferably at least about90%,andmost preferably at least about 95% ormore homologous to the nucleotide sequence, or fragment thereof, can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon the sequence of the one or more biomarkers listed in Table I and the Examples, orfragment thereof or the homologous nucleotide sequence. For example, mRNA can be isolated from muscle cells (i.e., by the uanidinium-thiocvanatie extraction procedure of Chirgwin e/ aL (1979) Bocheinhry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (ie Moloney MLV reverse transcriptase, avadable from GibcoBRL, Bethesda MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL), Synthetic oligonucleotide primers for PCR amplification can be designed according to well-known methods in theart. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNAas I template andappropriate oigonueleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into aniappropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to the nucleotide sequence of one or more biomarkers listed inTable I and the Examples can be prepared by standard synthetic techniques, .e, using au automated DNA synthesizer, Probes based on the nucleoide sequences of one or more bionarkers listed in Table I and the Examples can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a labelgroup attachedthereto, t e, the label group can bea radioisotope,a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used.as a part of a diagnostic test kit foridntifying cells or tissue which express one or more biomarkers listed in Table I and the Examples, such as by measuring a level of nucleic acid in a sample of cells from a subject, ce- detecting mRNA levels of one or more bionarkers listed in Table I and the Examples. Nucleic acid molecules encoding proteins corresponding to one or more biomarkers listed in Table I and the Examples from different species are alsocontemplated.For example, rat or monkeyDNA can be identified based on the nucleotide sequence ofa human and/or mouse sequenceandsuchsequencesare well knownin thwart. in one embodiment, the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of one or more biomarkers listed in Table I and the Examples, such that the protein or portion thereof modulates (vg, enhance), one or more of the following biologicalacnvities:a) binding to the biomarker; b) modulating the copy number of the biomarker; c) modulating the expression level of the biomarker; and d) modulating the activity level of the biomarker As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a. minUimum number of identical or equivalent (eg. an amino acid residue which has a similar side chain asan amino acid residue in one or more biomarkerslisted in Table I and the Examples, or fragment thereof) aminoacid residues toan amino acid sequence of the biomarker, or fragment thereof, such that the protein or portion thereofmodulates (e.g., enhance) one or more of the following biological activities: a) binding to the biomarker; b) modulating the copy number of the biomarker; c) modulating the expression level of the biomarker; and d) modulating the activity level of the biomarker, In another embodiment, the protein is at leasi about 50%, preferably ai least abou 60%, more preferably at least about 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%,9%, 99% or more homologous to the entire amino acid sequence of the biomarker, or a fmgment thereof. Portions of proteins encoded by nucleic acid molecules of the one or more biomarkers listed in'Tableland the Examples are preferably biologically active portions of the protein. As used herein, the term "biologicallyactive portion" of one ormore biomarkers listed in Table I and the Examples is intended to include a portion, etg, a domainfmotif, ihaihas one or mnor of the biological activities of the full-length protein. Standard bindingassays, eag inmmunoprecipitations and yeast tvo-hybrid assays, as described herein, or functional assays, e.g, RNAi or overexpression experiments, can be performed to deterine the ability of the protein or a biologically active fragment thereof to maintain a biological activity of tie full-length protein. The invention further encompasses nucleicacid molecules that differ from the nucleotide sequence of the one or more biornarkers listed in Table 1 and the Examples, or fragment thereof due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence, ortfragmentthereof In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding protein having anamio acid sequence of one ormore biomarkers listed in Table I and the Examples, or fragment thereof, or aprotein having an amino acid sequence which is at least about 70%, 75%, 80%, 85%, 901% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence of the one or more biomarkers listed in Table 1 and the Examples, or fragment thereof In another embodiment, a nucleic acid encoding a polypeptide consists of nucleic acidsequence encodinga portion of a full-length fragment of interest that is less than 195, 190, 5 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length. It will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the ainno acid sequnces of the one or more bioiarkers listed in Table I and the Examples may exist within population a maimmalan and/or human population). Such genetic polymrphisms may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding one or more biomarkers listed in Table I and the Examples, preferably a manmalian, eg, human, protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the one or more biomarkers listed in Table 1 and the Examples Anyand all such nucleotide variations and resulting-amoacidpolyiorphisms in the one or more bionarkers listed in Table I and the Examples that are the result of natural alelic variation and that do not after the functional activity of the one or more biomarkers listed in Table I and the Examples are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding one or more biomarkers listed in Table I and the Examples from other species. In addition to naturally-occurringallelic variants of the one ormore bionarkers listed in Table I and the Examples sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence, or fragment thereof. thereby leading to changes in the amino acid sequence of the encoded one ormore biomarkers listed in Table 1 and the.Examples, withoutaltering thefunctional ability of the one or more biomarkers listed in Table I and the Examples. For example, nucleotide substitutions leading to amino acid substitutions at non-ssntial" aino acid.resides can be made in the sequence, or fragment thereof. A non-essential" amino acid.residue is a residue that can be altered from the wild-type sequence of the one or more biomarkers listedin TableI and theExmIpleS without altering. the activity of the one ormore biomarkers listed in Table I and the Examples, whereas an "essential" amino acid. residue is required for the activity of the one or nore biomarkers listed in Table 1 and the Examples. Other amino acid residues, however, (cg, those that are not conserved or onlysemi-conserved between mouse and human) may not be essential for activity and thusare likely to be amenable to alteration without altering theactivity of the one or more biomarkers listed in Table I and the Examples. The term "sequence identity or homology" refers to the sequence similarity between two polypeptide molecules orbetween two nucleic acid molecules. Whenapositionin both of the two compared sequences is occupied by the same base or amino acid. monomer subunit, e.g.,if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identicalat that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shed by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in twosequencesare the same then the two sequences are 60% homologous or have 60% sequence identity. By way ofexample, the DNA sequences ATGCC and. TATGGC share 50% homology or sequence identity. Generally, a comparison is madewhen two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g those arising from, from deletions or insertions in one of the sequences are counted as nismatches. The comparison of sequencesand determination of percent homology between two sequences can be accomplished using a mathematical algorithm. Preferably, the alignment can be performed using the Clustal Method, Multiple alignment parameters include GAP Penalty =10, Gap Length Penalty = 10. For DNA alignments, the pairwisealignment parameters can be Htuple=2, Gap penalv=5. Window=4, and Diagonal saved=4. For protein aignmints the pairwise alignment parameters can be Ktuple=1, Gap penalty=:3, Window=, aid Diagonals Saved=5. In a preferred embodiment, the percent identity bevween twoaminoacid sequences is determined using the Needleman and Wunsch (J Mo Biol (48):444-453(1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1. 2 3, 4, 5, or 6 In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available onhnu), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of

1, 2,3, 4, 5, or 6. In another embodiment,the percent identity between twoamino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0) availablee onlie), using aPAMl 20 weight residue table, a gap length penalty of 12 anda gap penalty of 4. An isolated nucleic acid molecules ncoding a protein homologoustoone or more biomarkers listed in Table I and the Examples, or fragment thereof, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence, or fragment thereof, or a homologous nucleotide sequence such that one ormore amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, suchas site-directed rnutagenesisand PCR-mediated mutagenesis, Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A"conservative amino acid substitution" is one in which the aminoacid residue is replaced with an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include aminoacids with basic side chains (c.gf lysine, rgie histidine), acidic side chains (e.g.asparicacid, glutamic acid), uncharged polar side chains (e.g, glyine, asparagine, glutanine, serine, threonine, tyrosine. cystei), nonpolar side chains (eg, alanine, valine, leuci, isoleuci, proline, phenyialanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine) and.aromatic side chains (eg, tyrosine, phlenlanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in one or more biomarkers listed in Table 1 and the Examples is preferably replaced with another amino acid residue from the same side chain family, Alternatively, in another embodiment, mutations can be introduced randomlyalong all or part of the coding sequence of the one ormore biomarkers listed in Table I and the Examples, such as by saturation imutagenesis, and the resultant mutants can be screened for an activity described herein to identity mutants that retain desired activity. Following mutagenesis, the encoded protein can be expressed recombinantly according to well-known methods in theart and theactivity of the protein can be determined using, for example, assays described herein. The levels of one or more biomarkers listed in Table I and the Examples levels may be assessed by any of a wide variety of well-knownmethods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assys, nucleicacid y bridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods, In preferred embodiments, the levelsof one or more biomarkers listed inTable 1 and the Examples levels areascertained by measuring gene transcript (e,g., mRNA), by a measure of the quantity of translated protein, or by ameasure of gene product activity. Expression levels can be monitored inma varey of ways including by detecting mRNA levels,protein levels, or protein activity any of which can be measured using standard techniques Detection can involve quantification of the level of gene expression (eg, genoic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitativeassessment of the level of gene expression, in particular in comparison with a control level The type of level being detected will be clear from the context, In a particular embodiment, the mRNA expression level can be determined. both by in situ and by in vitro fonnats in a biological sample using methods known in theart. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject, Many expression detection methods use isolated RNA. For in vitromethods, any RNA isolation technique that does not select against the isolation ofmRN A can be utilized for the purification of RNA from cells (see,ea Ausubel el alt ed, Current Protocols in Mo/ecuarBiogy, John Wiley & Sons,Ncv York 198-1999), Additionally, large numbers of tissue samples can readily be processed using techniqueswellknown to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155. The isolated mRNA can be used in hybridization oramplification assays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic methodfor the detection of mRNA levels involves contacting the isolated mRNA with nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-lenagth cDNA, ora portion thereof such as an oligonucleotide of at least 7, 15, 30,50, 100,250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genormic DNA encoding one ormore biomarkers listed inTable I and the Examples, Other suitable probes for use in the diagnostic assays of the invention are described herein. Hvbridization ofan mRNA with the probe indicates that one or more biomarkers listed in Table I and the Examples is being expressed In oneformat, themRNA is immobilized on a solid surfaceand contacted with a probe, for example by nning the isolated mRNA onan agarose gel and transferring the mRNA from thegel to a membrane, suchas nitrocellulose, Inan alternative format, the probes) are iunobilized on a solid surface and the nRNA is contacted with the probe(s), for example, in a gne chip array, e.g., an Affyinetrix" gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of theOne or more biomarkers listed in Table 1 and the ExamplesmRNA expression levels. An alternative method for determining mRNA expression level in sample involves the process of nucleic acid amplification, e.g, by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proca A ca. USA, 88189-193), self-susined sequencereplication (Guateli et al. 1990, o .at Aad. Sci.USA 87:1374-187), transcriptional anplification system (Kwohi 1989troal.. Acad. Sci, UA 86:1173-1177), QBeta Replicase (Lizardi Ca,198 i/cnlog' 6:1197). rolling circle replication (Lizardi et al, U.S Patent No. 5,854,033) orany other nucleic acid amiplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleicacid molecules if such molecules arepresent in very low numbers. As used herein, amplification primersare defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain short region in between. In general,amplificationprimers are from about 10 to 30 nucleotides in length and flank a.region fronabout 50 to 200 nucleotides in length. Under appropriate conditionsand with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotidesequence flanked by the primers. For in situ methods, mRNA does not need to be isolated from the cells prior to detection. Ia suchmethods, a cell or tissue sample is preparedprocessed using known histological methods. The sample is then immobilized ona support, typically a glassslide, and then contacted with a probe that can hybridize to the One or more biomarkers listed in Table 1 and the Examples mRNA. Asan alternative to making determinations based on the absolute expression level, determinations may be based on the normalized expression level of one or more biomarkers listed in Table I and the Examples. Expression levels are normalized correcting the absolute expression level by comparing its expression to the expression of a nonbiomarker gene,e.g., a housekeeping gene that's constitutively expressed. Suitable genesfor normalization include housekeeping genes such as the acting gene, orepithelial cell-specific genes. This normalization allows the comparison of the expression level in onesample, eg a subject sample to another sample,e.anormalsampleorbetweensamplesfrom different sources. The level or activity of a protein corresponding to one ormore biomarkers listed in Table I and theExamples can also be detected and/or quantified bydetecting or quantifying the expressed polypeptide, The polypeptide can be detected and quantified by any of a number of means well known to those of skill in thwart. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chrimiatography (TLC),hperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radiiimmuntassay(RIA), enzyne-linked immunosorbent assays (ELISAs), imiunofluorescent assays, Westeru blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the biomarker of interest The present invention further provides soluble, purified and/or isolated polypeptide fomns of one or more biomarkers listed in Table 1 and. the Examples, or fragments thereof. In addition, it is to be understood that any and all attributes of the polypeptides described herein, such as percentage identities, polypeptide lengths, polypeptide fragments, biological activities, antibodies, etc. can be combined in any order or combination with respect to any biomarker listed inTable I and the Examples and combinations thereof. In one aspect, a polypeptide may comprise a full-length amino acid sequence correspondingto one or more biomarkers listed in Table I and the Examples or afill length amino acid. sequence with I to about 20 conservative amino acid substitutions. An anino acid sequence of any described herein can also be at least 50, 55, 60, 65, 70, 75,80, 85, 90, 91 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identical to the full-length sequence of one or more biomarkers listed in Table I and the Examples, which is either described herein, well known in the art, or a fragment thereof In another aspect, thepresent invention contemplates a composition comprising an isolated polypeptide corresponding to one or more biomarkers listed in Table I and the Examples polypeptide and less than about 25%, oraternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides. The present invention further provides compositions related to producing detecting characterizing, or modulating the level or activity ofsuch polypcptides, or fragment thereof such as nucleicacids, vectors, host cells, and the like. Such compositions may serve as compounds that modulate the expressionand/or activity ofone or more biomarkers listed in Table I and theExamples. For example, anti-PD-L1 antibodies thatmay bind specifically to PD-L. or soluble PD- Ican be used to reduce soluble PD-L (i.e., both forms of PD-LI containan extracellular domain typically targeted by antibodies) and thereby a)stop the titration of such therapeuticaentsfrom binding to membrane-bound forms of PD-1. and/or b) inhibit the inhibition of immunological responses promoted by the soluble forms oFPD-L1, An isolated polypeptide or a fragment thereof(ora nucleicacid encoding such a polypeptide) corresponding to one or more biomarkers of the invention, including the biomarkers listed in Table I and the Examples or fragments thereof, can be used as an imunurriogen to genermtantbodis that bind to said immnogen, using standard techniques for polyclonal and monoclonal antibody preparation according to well-known methods in the art An antigenic peptide comprises at least 8 amino acid residues and encompasses an epitope present in the respective ftull length molecule such that an antibody raised against the peptide forms a specific immunecomplex with the respectivefdUll length molecule Preferably thle atigenic peptide comprises at least 10 amino acid residues. In one embodiment such epitopes can be specific fora given polypeptide molecule from one species, suchas mouse or human (ie,an antigenic peptide that spans a region of the polypeptide molecule that is not conserved across species is used as immunogen; such non conserved residues can be determined usingan alignment suchas that provided herein) For example, a polypeptide inmnogen typically is used to prepare antibodies by iinnmizinga suitable subject (e.g rabbit, goat, mouse or other mammal) with the iinnmogen. An appropriate iminogenic preparation can contain, for example, a recombinantly expressed or chemicallysynthesized. molecule or fragment thereof to which the itumnne response is to be generated. The preparation can further include an adjuvant., such as Freunds complete or incomplete adjuvant, orsimilarimnnosimatory agent.

nnmnization of a suitable subject with an immunogenic preparation induces a polyclonal antibody response to the antigenic peptide contained therein. Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide immunogen. The polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked inunuosorbent assay (ELISA) using irnobilized polypeptide If desired, the antibody directed against the antigen can be Isolated from the mammal (e.g frorn the blood.) and further purified by Nell-known techniques, such as protein A chromatography, to obtain the IgG fraction. At an appropriate time after muniation e.giwhen the antibody titers are highest. antibody-producingcells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique(originally described by Kohlerand Milsicin (1975) Nature256:495-497)(see also Brown et (1981) J hnmuni w 127:539-46; Browio e al (1980) Biol Chem. 255:4980-83; Yeh e/ at (1976) Proc. Nal Acad. Si. 76:2927-31; Yeh et al. (1982)nt.1 Cancer29:269-5 thimore recent human B cell hybridoa technique(Kobor et al (1983) immunolt tda 4:72), the EBV-hybridoma technique (Cole et a L (1985) MAonodona Antibodies and an herap, Alan R. Liss, Ic, pp. 77-96) or triona techniques. The technology for producing monoclonalantibody hybridonas is well known (see generally Kenneth, R.H in MonoceonalAntibodies: A New Dinension i Biologica/na s Plenum Publishing Corp, New York,New York (1980); Leer, E A. (1981) Ye J, Biol Med. 54:387-402; Gofter, M L. et at (1977) Sonatic Ce Genet.231-36). Briefly, an innortal cell line

(typically a myclonia) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptide antigen, referablyspecifically. Any of the many well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating amonoclonal antibody against one or more bionarkers of the inventio, including the biomarkers listed inTable 1 and the Examples, or a fragment thereof (see, e. Galfre, G. et al (1977)Nare 266:550 52;Ocferta(1977)supra;Lemner(198)suprKenneth(1980)supra).Moreoverthe ordinary skilled worker will appreciate that there emany Variations of such methods which also would be useful. Typically, the inimmortal cell line (e.g, a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, urine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of tih present invention with an immortalized mouse cell line. Preferred immortal Cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, amnopterin and thymidine ("HAT medium"). Any of a number of neloma cell lies can beused as a fusion partneraccording to standard techniques, eg the P3-NS1I1 -Ag4-1, P3-x63-Ag8.653 r Sp0O-Agl4 myloma lines, These myelona lines re available from the American Type Culture Collection (ATCC), Rockville, MD. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usiny polyethylene glycol ("PEG"). Ilybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myCloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibdies that bind a given polypeptide, e.g, using a standard ELISA assay, As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal specific for one ofthc above described polypeptides can beidentifiedand isolated by screening a recombinant conbinaorial immunoglobulin libry (e.g, an antibody phage display library) with the appropriate polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g, the Pharmacia Recombinam Phage AntibodS' m Catalog No, 27-9400-01; and the Stratagene

Swf24PT' MPhage Display Kit, Catalog No 240612). Additionally, examples of methods and reagents particularly amenable for use in gnerating and screening an antibody display library can be found in, for example, Ladner et al US, Patent No. 5;223,409; Kang et aL internationalPubliatoNo. WO 9218619; Dower et al International Publication No. WO 91/17271; Winter el al International Publication WO 9220791; Markland e at International Publicaton No. WO 92/15679; Breiting et a International Publication WO 93/01288; McCafferty er ax international Publication No. WO 92/01047: Garrard et a, International Publication No WO 92/09690; Ladner etal International Publication No. WO 90/02809; Fuchs el aL (1991) Bioechnology 9:1369-1372; Hay et a, (1992) Hum. Antibod Hybrhdomas3:81-85; Huse eia (1989))Science 246:1275-1281 Gyriffiths et al (1993) EMBO Biol 226:889-896; Clarkson 12:725-734; Hakins et aL (I992)JMR et al (1991)ANaure 352:624-628; Gram et al (1992) Pon, Nat Acad Sci USA 893576

3580; Garrard et ad (1991) Bitechnology (NY9:1373-1377; Hoogenboomieta (1991) Nwuclei Acids Res. 19:4133-4137; Barbas et aL (1991) ProcNa lAcad.Sdc.USA 88:7978 7982: and McCafferty etL (1990) \aure 348:552-554, Additionaliy, recombinant polypeptide antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, Le within ihe scope oftheinvention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in theart, for example using methods describedin Robinson el al International Patend Publication PCT/US86'02269; Akira et al European Patent Application 184,187; Taniguchi, M, European Patent Application 171,496; Morrison el aL European.Patent Application 173,494; Neubeerr et al PCT Application WO 86;01533; Cabilly et al U.S. PatentNo. 4,816,567; Cabilly elaVEuropean Patent Application 125,023; Better e/ aL (1988) Science 240:1041-1043; Liet a? (1987) Proc. NYat Acad Sci. USA 84:3439-3443;, Liu o a/.l 987) Immund 139:3521-3526; Sun et ad (1987) ProcNaLcad S 84:214-21 SNishimura eatd (987) Cancer.Res 47:999-1005; Wood et al (1985),ature 314:446-449; Shaw et ad (1988)1 N. adtL erInst. 80:1553 1559); Mrrison, S. L (1985) Scce29:1202-1207; iet1a286) ioechniques 4:214; Winter U.S. Patent 5,225,539; Joneseta (1986) Nature321:5525;ehoeyant af (1988) ScienceW239:1 534; and Beidler et a (1988)J hnmunoL 141:4053-4060, inaddionhumizdanibodis can bemade according to standard protocols such as those disclosds In US. Patent 5,565,332. In anotherembodiment, antibody chains or specific binding pairmembers can be produced by recombination between vectors comprising nucleic acid molecules encodinga fusion ofa polypeptide chain ofa specific binding pair member and a coinponent of a replicable generic display package and vectors containing nucleicacid molecules encoding a second polypeptide chain of a single binding pair member usingtcchniqWs known in the art, e., as described in U.S. Patents 5,565,332, 5,871,907,or5,733,743. The use ofintracellular antibodiesto inhibitproteintfunction in a cell is also known inthe art (see eg. Carlson, J. R, (1988)M Cell Bio 8:2638-2646; Biocca, S. et aI (1990) EMBO J.9:101-108; Werge, T. M. et al (1990)KKS Le 274:193-198; Carlson, J. R- (1993) Proc. Sc St4ad 90:27-428; Marasco, W. A. ct al. (1993)Proc. NA adSci USA 90:7889-7893; Biocca, S, et at (1994) Biotechnolovn(N}I12:396-399; Chn, S-Y et aL (1994)Hum. (ene Ier. 5:595-601; Duan,LetiaL (1994) Proc. N c Sci. USA 91:5075-5079; Chen, S-Y. era/.(1994)

Proc, NatL Acad Sc. USA 91:5932-5936; Beerli, R. Rer a (1994) J Biol Chem, 269:23931-23936;Beeri, R. R, et al (1994) Biochem Biophys. Res. Common. 204666 672; Mhashilkar, A. M. et al (I995) EXhO J. 14:1542-1551 Richardson, J, , et al (1995) Proc. NatlAcead Sei. USA 923137-3141; PCT Publication No. WO 94/02610 by Marasco et alt; and PCT Publication No. WO 95/03832 by Duan elta. Additionally flly human antibodies could be made against biomarkers of the invention, including the biomarkers listed in Table I and the Examples, or fragments thereof FulyIxhuman antibodies can be made in nice theatre transgenicforhuman imnunoglobulingenes, ecg, according to Hogan, et ad- "Manipulating the Mouse Embryo A Laboratory Manuel," Cold Spring Harbor Laboratory. Briefly, transgenic miceare immunized with purified imunogen. Spleen cells are harvested andfused to myeloma cells to produce hybridomas. Hybridomas are selected based on theirabilityto produce antibodies which bind to the immunogen, Fully human antibodies would reduce the mimunogenicity of suchantibodies in a human. In one embodiment, anantibody for use in the instant invention isa bispecific antibody. A bispecificantibody has binding sites fortwo different antigens within a single antibody polypeptide. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomias are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecificantibodies produced by a hybrid hybridoma ora trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al (1985) Nature 314:628, and Perez e/ al (1985)Nature 316:354) and hybridoma technology (Starz and Bevan (1986) Proc. Na. Acad. Sei. USA, 83:1453, and Star and Bevan (1986) ImmunoL Toayi 7:241). Bispecific antibodies are also described in U,S. Patent 5,959,084. Fragments of bispecific antibodies are described in US. Patent 5,79s8229. Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling bothantibodies, They can also be generated by chemical or genetic conijugation of completeiImunoglobulin chains or portions thereof such as Fab and. Fv sequences. The antibody component can bind to a polypeptide or a fragment thereof of one or more biomarkers ofthe invention, including one or more biomarkers listed in Table 1 and the Examples, or a fragment thereof. In one embodiment,the bispecifieantibody could specifically bind to both a polypeptide or a fragmnent thereofand its natural binding partner(s) or a fragments) thereof In another aspect of this invention, peptides or peptide mimetics can be used to antagonize or promote theactivity of one or more biomarkers of the invention, including one or more biomarkers listed inTable I and the Examples, ora fragment(s) thereof In one embodiment, variants of one or more biomarkers listed in Table 1 and the Examples which function as a modulatingaent for the respective fill length protein, can be identified by screening combinatorial libraries of mutants, eg truncationmutantsforantagonist activity. In one embodiment, a varlegated library of variants is generated by combinatorial mutagcnesis at the nucleic acid level and is encoded by a variegated gene library, A variegated library of variants can be produced, for instance, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential polypeptide sequences is expressibleas individual polypeptides containing the set of polypeptide sequences therein- There are a variety of methods which can be used to produce libraries of polypeptide variants from a degenerate oligonucleotide sequence Cberical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer,and the synthetic gene then ligatedinto an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of thequences encoding the desired set of potential polypeptide sequences. Methods for synthesizing generate oligonucleotides are known inthieart (see, e g, Narang, S. A. (1983) Tetrahedron 39:3 itakura et al (1984) Annu. Rev- Biochem 53:323; Itakura e/ al (1984) Scien-c 198:1056 Ike et al (1983)Nuleic Acid Res 1.1:477. In addition, libraries of fragments of a polypeptide coding sequence can he used to generate a variegated population of polypeptide Fragments for screening and subsequent selection of variantsof aogivenpolypeptide. Inonembodiment,alibraryofcoding sequence fragments can be generated by treating a double stranded PCR fragment of a polypeptide coding sequence with a nuclease under conditions wherein nicking occurs only about once per polypeptide, denaturing the double stranded DNA, renaturing the DNA to form double stranded. DNA which can include sense/antisense pairs from different nicked. products, removing single stranded portions from reformed duplexes by treatment with SI nuclease,and ligating theresulting fraginent library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the polypeptide.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of polypeptides. The most widely used techniques, whichare amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected, Recursive ensemble mutagenesis (REM), atechniquewhichenhances the frequency of functional mutants in the libraries, can be used in combination with the screeningassays to identify variants of interest (Arkin and Youvan (1992) Proc. Nad. Acad Sc .USA 89:7811-7815 Delagraveet a (1993) Protein ng.6'3327-331). In one embodiment, cell based assays can be exploited toanalyze a variegated polypeptide library. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes one or more biomarkers of theinvention, including one ormore biomarkers listed in Table I and the Examples, or afragment thereof The transfected cells are then cultured such that thefull length polypeptide and a particular mutant polypeptide are produced and the effect of expression of the mutant on the full length polypeptide activity in cell supernatants can be detectede.g., by any of a number of functional assays. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of full length polypeptide activity, and the individual clones further characterized. Systematic substitution of one or more amino acids ofa polypeptide amino acid sequence witha D-aminoacid of the same type (e.g, D-lysine in place ofL-lysine) can be used to generate more stable peptides. In addition, constrained peptides comprising a polypeptide amino acid sequence of interest or a subsantially Identical sequence variation can be generated by methods known in the art (Rizo and Gierasch (1992) Anna Rev. Biochem, 61:387, incorporated herein by reference); for example, by adding internal cvsteine residues capable of forming intramolecular disulfide bridges vhich cyclize the peptide, The amino acid sequences disclosed herein will enable those of skill in the art to produce polypeptides corresponding peptide sequences and sequence variants thereof

Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleondes encoding the peptide sequence, frequently as part of a larger polypeptide. Alternatively, such peptides can be synthesized by chemical methods. Methodsfor expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in itro translation are well known in the art and are described further in Mariatis et a M'folecular Cloning: A LabonaoryManual (1989) 2nd Ed, Cold Spring Harbor, NY,; Berger and Kinmel Methods in Enzymology Volume 152, Guide to Molecular Cloning Techniques (I987), Academic Press, Inc. ,SanDiego, Calif. Merrifield, 5 (1969)/Am. Chemn.Soc. 91:501; Chaiken I. M. (1981) CRC Cr ev. Biochcm.I1: 255; Kaiser et a (1989) Science 243187; Merrifield, B, (1986) Science 232:342; Kent, S. B. H. (1988)Annu. Rev. BHochent 57:957; and Offord, R. E. (1980) Semisn/hetic mteins, Wiley Publishing, whichare incorporated herein by reference). Peptides can be produced, typically by direct chemical synthesis. Pptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terninus- In certain preferred embodiments, either the carboxy terminus or the anino-terninus, or both, are chemically modified, Theinost common modifications of the terminalamino and carboxyl groups are acetylation and amidation, respectively, Amino-terminal modifications such as acylationt (eg acetylation) or alkylation (e.g, methylation) and carboxy-terminal-modifications such as anidation, as well as other terminal modifications, including cyclization,canbeincorporatedintovarous embodiments of the invention. Certain amio-terminal and/or carboxy-terinnal modificationsandorpeptide extensions to the core sequence can provideadvantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. Peptides disclosed herein can be used therapeutically to treat disease, e.g, by altering costimulation in a patient Peptidomimtis (Fauchere, J (1986) Ad, D Res. 15:29; Veberand Freidinger (1985) TINS p,392; and Evans et aL (1987)J tVed Chen, 30:1229, which are incorporated herin by reference) are usually developed with the aid of computerized molecular modeling. Peptide mimetics thatare structurallysimilar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect, Generally,peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: CH2N- -CH2S,-C2-CH2-, ~CH=CH- (cis and trans), -COCH2-, ~CH(OH)CH2, and C12SO-, by methods known M the art and further described in the following references: Spatola, A- F -in"Chemistrv andBiochemisny ofAmino Acida Pptids,andProtins" Weinstein, B- ed, Marcel Dekker, Nwv York, p. 267 (1983); Spatola, A, F, Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifeations" (general reviw) Morley, J. S. (19W) Tena harm. I. pp. 463-468 (general review); Hudson, D- et a/ (1979) In.J. Pept Prot. Res. 14:177~185 (-CH2NH CH2H2)Spatola, A1F et -d, (1986) Lie Sci. 38:1243-1249 (-(H2-S)l Hatn, M. M. (1982)JC SocPrkinTans, f 307-314 CH-CH, cis and trans), Ahnquist, R. G. et aL (190) JMeld Chem 23:1392 1398 (-COCH2-); Jennings-White, C. et al (1982) TerahedronLett.23:2533 (-COCH2-); SzelkeM et aL European Appin, EP 45665 (1982) CA: 97:39405 (1982)(~CH(OH)CH2 )Holladay, M. W. et al- (1983) a o Lent. (1983)24:4401-4404 (-C(OH)CH2); and Hruby, V. J (1982) LieScI. (1982) 31:189-199 (-CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is -C1-2NIH-I Such peptide mimetics may have significant advantages over polypeptide embodiments, inchiding, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-lifeabsoirion, potency, efficacy, etc.). altered specificity e.g.a broad-spectnum of biological activities), reduced antigenicity',and others, Labeng of pptidomiletics usually involves covalent attachment of one or more labels, directly or through a spacer (eg an amid group), to non-interfering position(s) on the peptidonimetic thatare predicted by quantitative structure-activitv data and/or molecular modeling. Suchnon-inerferingpositions generally are positions that do not form direct contacts with the macropolypeptides(s) to which the peptidomietic binds to produce the therapeutic effect. Derivitization (e.g, labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. Also encompassed by the present invention are small molecules which can modulate (either enhance or inhibit) interactions, e.g, between biomarkers listed in Table 1 and the Examplesand theirnatural binding partners, or inhibitactivity. Thesmall molecules of the presentivention can be obtained usingany ofthenumerousaproaches in combinatorial library methods known in the art, including: spatially addressable parallel solid phase or solution phase libraries; synthetic library methods reqtiring deconvolution the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. (Lam, K, S. (1997) Anicancer Drug Des, 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt etcil (1993) Prtoc.NatLvAcad Sci. 90:609; Erb elet at(1994) ProcA w. Acad Si. S USA 91:11422; Zuckernann et at (1994)J Med. Chem. 37:2678 Cho e/ a? (1993)Science 261:1303; Carrell ei a. (994)Agew. Ciem.mIuT n. 33:2059; Carell eta. (1994) Anget. Chem. Fn.£ ngL,1 33:2061; and in Gallop et al (1994),1( Chem. 37:1233. Libraries of compounds can be presented in solution (eg. Houghten (1992) Biotechniques 13:412-42V) or on beads (Lam(1991).Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et at (1992) PrcatAcad Sc. USA 891865-1869) or on phage (Scott and Strh (n. (1Scc249:386-390);(Dvli(1990)Scince 249:404-406) (Cwirla e/ at (1990) Proc. tL AecwSi.`USA 87:6378-382); (Felici (1991).!Mo Biol. 222:301 310);(Ladnersuprt.). Compounds can be screened in cell based or non-cell based assays Compounds can be screeed in pools (e.g.,multiple compounds in each testing sample) or asindividual compounds. The invention also relates to chimeric or fusion proteins of the biomarkers of the invention, including the biomarkers listed in Table 1 and the Examples, or fragments thereof As used herein, a "chineric protein" or "fiusion protein" comprises one or more biomarkers of the inventioni, Including one or more biomarkers listed in Table I and the Examples, or afragment thereof operatively linked to another polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the respective biomarker. In a preferred embodiment, the fusion protein comprises at least one biologicallyactive portion of one or more biomarkers of the invention. including one or more biomarkers listed in Table I and the Examples, or fragments thereof Within the fusion protein, the term "operatively linked" is intended to indicate that the biomarker sequences and the non-biomarker sequences are fused in-frame to each other in such a way as to preserve functions exhibited when expressed independently of the fusion. The "another" sequences can be fused to the N-terminus or C-terminus of the biomarker sequences, respectively, Such a fusion protein can beproduced by recombinant expression of a nucleotide sequence encoding the first peptide and a nucleotide sequence encoding the second peptide

The second peptide may optionally correspond to a moiety thatalters the solubility, affinity, stability or valency of the firstpeptide, forexample, an rimnmunoglobulin constant region. hi another preferred embodiment, the first peptide consists of a portion of a biologically active molecule (egthe extracellular portion of the polypeptide or the ligand binding portion), The second peptide can include an immunoglobulin constant region, for example, a humanC I domain r Cy4 domain (e.g., the hinge, CH2 and CH3 regions of humanIgCvy

1, or human IgCy4, see eg., Capon e l U.S Patents 5,116,964; 5,580,756; 5,844,t95and the like, incorporate here by reference), Such cnstant regionsmay retain regions which mediate effector function (, Fe receptor binding) or may be altered to reduceeffector function. A resulting fusion protein may havealtered solubility, binding affinity, stability and/or valency (ix, the number of binding sites available per polypeptide) as compared to the independently expressed first peptide, and may increase the efficiency of protein purification. Fusion proteins and peptides produced by recombinant techniques can be secreted and isolatedfrom a mixture of cellsand medium containing the protein or peptide Alternatively, the protein or peptide can be retained vtoplasmically and the cells harvested. lysed and the protein isolated. A cell culture Ntyically includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides, Techniques for transfecting host cells and purifying proteins and peptides are known in the art. Preferably, a fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frane in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for Igation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation, In another enbodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs betwven two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols inMolecularBioogv,eds. Ausubel et L John Wiley & Sons: 1992).

Inanother embodiment, the fusion protein contains aheterolooussignalsequence at its N-terminus. In certain host cells(eigmammalian host cells), expressionand/or secretion of a polypeptide can be increased through use of a heterologous signal sequence. The fusion proteins of the invention can be used as immunogens to produce antibodies inasubject. Such antibodies may be used to purify the respectivenatural polypeptides from which the fusion proteinsweregertd or in screening assays to identifypolypeptides which inhibit the interactions between one or more biomarkers polypeptide or afragment thereofand its natural binding partner(s) ora fragment(s) thereof Also provided herein are compositions comprising one or more nucleic acids comprising or capable of expressingat least 1, 2_3, 4, 5, 10, 20 or more small nucleic acids or antisense oligonucleotidesor derivatives thereof; Wherein said small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell specifically hybridize (eg, bind) under cellular conditions, with cellular nucleicacids (e.g, small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miRNA binding site, a variantand/or functional variant thereof, cellular mRNAs ora fragments thereof)- In one enbodinent, expressionof the small nucleic acids or anisense oligonucleoides or derivatives thereof in a cell canenhance orupregulate one or more biological activities associated with the corresponding wildtype, naturily occurring, or synthetic small nucleic acids. Inanother embodiment, expression of the small nucleic acids orantisense oligonuclcotidesorderivatives thereofin a cell can inhibitexpression orbioloicalictivit ofcellular nucleic acids and/or proteins, c" by inhibiting transcription translation and/or small nucleic acid processing of, for example, one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples, or fragment(s) thereof. In one embodiment, the small nucleic acids or antisense oligoncleotides or derivatives thereof are small RNAs (e.g., microRNAs) or complements of small RNAs. In another embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof can be single or double stranded and are at least six nucleotides in length and are lessthanabout1000900,800, 700, 600, 500 400, 300 200, 100, 50, 40, 30, 25, 24, 23, 22 2120, 19, 18, 17, 16, 15, or 10nuleotidesin length. In another embodiment, a composition may comprise a library of nucleic acids comprising or capable of expressing small nucleic acids or anisense oligonucleoides or derivatives therCof, or pools ofsaid small nucleic acids or aitisense oligonucleotides or derivatives theren£ A pool of nucleie acids may comprise about 2-5, 5-10, 10-20, 10-30 or niore nucleic acids comprising or capable of expressing small nucleic acids or antisense oligonucleotides or derivatives thereof Inoneembodiment, binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions inlthe major groove of the double helix. In general, "antisense" refers to the range of techniques generally employed in the art, and includes any process that relics on specific binding to oligonucleotide sequences. It is well known in the art that modifications can be made to the sequence of a miRNA or a pre-miRNA without disrupting miRNA activity. As used herein, the term "functional varianC" of a niRNA sequence refers to an oligonucleotide sequence that varies from the natural miRNA sequence, but retains one ormore functional characteristics of the miRNA (e.g., cancer cell proliferation inhibition, induction of cancer cell apoposis, enhancementof cancer cell susceptibility to chemotherapeutic agents, specific miRNA target inhibition). In some embodiments, a functional variant of a miRNA sequence retains all of the functional characteristics of the miRNA. In certain embodiments, a finctional variant of a iRNA has a nucleobase sequence that is a least about 60%, 65%.70%, 75/0 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 964, 97%, 98% or 9%identicalto the iRNA or precursor thereof over a region of about 5, 6, 7, 87 9, 10, i1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases. or that the functional variant hbridizestothecomplementofthe miRNA or precursor thereof under stringent hybridizaonconditions. Accordingly, in certain embodiments the micleobase sequence of1a functional variant is capable of hybridizing to one or more target sequences of the miRNA. niRNAs and their corresponding stem-loop sequences described herein may be found in niRBase, an online searchable database of miRNA sequences andannotation, found on the world wide web atmirorna.sanerac.uk Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with inforiation on the location and sequence of the rnaturetiiRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), ad. may in someinstancesnlude the pre-miRNA and someflanking sequence from the presumed primary transcript, The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 100 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs, A sequence database release may resulting a variation of amature miRNA sequence, In some embodiments, miRNA sequences of the invention may be associated witha second RNA sequence that may be located on the same RNA molecule or on a separate RNA molecule as ti miRNA sequence. In such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence, which is at least partially complementary to the miRNA sequencemay be referred to as the complementary strand. The active and complementary strands are hybridized to create a double-stranded RNA that issimilar to naturally occurring miRNA precursor. TheiityofxaniRNAmaybe optimized by maximizinguUptake of the active strandand minimizing uptake of the complementary strand by the miRNA protein complex that regulates translation. This can be done through modification and/or design of the complcmcntary trand, In some embodiments, the complementary strand is modifiedso thata chemical groupother thanaphosphateorhydroxylatits -5terminus- Thepresence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of the active strand by the miRNA protein complex, The 5'modification can be any of a vriety of molecules known in the art, including NH NHCOCH and biotin In anotherembodiment, the uptake of the complementary strand by themiRNA pathway is reduced by incoporating nucleotides with sugar modifications in the first 2-6 nucleotides of theomplementy ustrand.itshould be noted. that such sugar modifications can be combined with the 5' terminal modifications described above to further enhance miRNA activities. In some embodiments, the complementarystrandis designed so that nucleotides in the 3' end of thecomplementary strand are not complementary to the active strand. This results in double-strandhybrid RNAs that are stable at the 3'end of theactive strand but relatively unstable at the 5'end of the active stand, This difference in stability enhances the uptake of the active strand bythe miRNA pathway, while reducing uptake of the complementary strand, thereby enhancingmRNA activity. Small nucleic acidand/or antisense constructs of the methods and compositions presented herein can be deliveredfor example, as an expression plasmid which, when transcribed inthe cell, produces RNA which is complementary to at least aunique portion of cellular nucleic acids (eg, small RNAs, mRNA, and/or genomic DNA). Alternatively, the small nucleic acidmolecules can produce RNAwhich encodes mRNA, miRNA, pre miRNA, pri-miRNA, miRNAanti-miRNA. or a miRNA binding site, or a variant thereof For example, selection of plasmids suitable for expressing the miRNAs, methods for inserting nucleic acid sequences into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng ei a! (2002), Molecular Cell 9:1327-13 Tuschl (2002)t, NaBiotechnol, 2(:446 448; Brunmelkamp era (2I2), Science296:550-553; Mivagishie a/ (2002), Nat Biotechnol. 20:497-500; Paddison et al (2002), Genes Dev. 16:948~958; Lee et al (2002), Nat. Biotechnolt 20:500-505 and Paul el aL (2002), Nat Biotechnol 20:505-508, the entire disclosure ofxwhich are herein incorporated by reference. Alternatively, small nucleic acids and/or antisense constructs are oligonucleotide probes that are generated ex vivo and which, when introduced into the cell results in hybridization with cellular nucleic acids. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant toendogenous nucleases, e.g, exonucleases and/or endonuleas and are therefore stable in vivo. Exemplary nuclic acid molecules for ise as small nucleic acids and/or antisense oligonucleotides arephphohramidate, phosphorhioate and mthylphosphontalgsof DNA (see also U.S. Patents 5,176,996; 5,264,564;and5,256,775) Additionally,generalapproachestoconstructingoligomers useful in antisense therapy have been reviewed, for example, by Van der Krol el a (1988) BioTechniques 6:958~976; and Stein etal (1988) Cancer Res 48:2659-2668. Antisense approaches may involve the design ofoligonucleotides(cither DNA or RNA) that are complementary to cellular nucleic acids (eg., complementary to biomarkers listed in Table I and ti Examples). Absolute complementarity is not required. In the case ofdouble-strandedantiensnucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree of complementarity and the length of theantisense nuclic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a nucleic acid (e.g, RNA) it may contain and still form a stable duplex (or triplex, as the case may be), One skilled in thwart canascertain a tolerable degree of mismatch by use of stadard procedures to determine the melting poit of the hybridized complex. Oligonuceoides that are complementary to the 5' end ofthe mRNA. eg., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R (1994) ature 372:333). Therefore, oligonucleotides complementary to either the 5' or T untranslated, non-coding regions of genes could be used in an antisense Ipproach toinhibit translation of endogenous mRNAs. Oligonucleotides complementary to the 5' untranslated region of theniRNA may include the complement of the AUG start codon, Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the methodsand compositions presented herein. Whetherdesignedto hybridize to the 5,3' orcodingregionof cellular mRNAs, small nucleic acids and/or antisense nucleicacids should be at least six nucleotides inlength, and can be less than about 1000, 900, 0, 700, 6W0,00, 400, 300, ,200,50, 40, 30, 25, 24, 23, 22,21,20, 100, 19, 18, 17, 16, 15, or 10 nucleotides in length, Regardless of the choice of target sequence, it is preferred that in vrostudies are first performed to quantitate the ability of theantisense oligoncleotide to inhibit gene expression. In one embodiment these studies utilize controls that distinguish between anusensegeneinhibitionand nonspecific biological effects of oligonucleotides, In another embodirent these studies compare levels of the target nucleic acid or protein with that of an. internal control nucleic acid or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide, ft ispreferred that the control oligonucleotide is of approximately the same length as the testoligonucletideand that the nucleotide sequence ofthe oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence. Small nucleic acids and/or antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double stranded. Small nucleic acids and/orantisense oligonuclcotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides (e.g. for targeting hostcell receptors), or agents facilitating transportacross the cell membrane(seeeg. Ietsingretat(1989) Proc. Nat.A cad. SciIU.A 86:-6553-6556; Lemaitre c al (1987) Proc. NatL Acad. Sci, 84:648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, eg, PCT Publication No. W089/10134, published April 25, 1988),hybridization-trigcred cleavage agents. (See,e~g, Krol el al (1988) BioTechniques 6:958976) orintercalatingagents. (See, etg, Zon (1988) Pharm. Res. 5:539-549). To this end. small nucleic acids and/or antisense oligonucleotides mav be coniugated to another molecule, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. Small nucleic acidsand/or antisense oligonucleotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5 fluorouracil, 5-bromouracil, 5-Chlorouracil. 5-iodouracil, hypoxanthine, xantine.4 acetylcvtosine, 5-(carboxyhydroxytiethyl) uracil, 5-carboxynethyninoiethyl-2 thiouridine, 5-carboxymethylaminomethyluracil, dihydrouiracl,btaDgaltoslqueosine, inosine, N6-isopentenyladenine, I-mCthylguaninI-, methylinosine, 2,2dimethylguaninc, 2-methyladenine, 2-methylguanine, 3-methyleytosine, 5-mlethyleytosine, N6~adenine, 7-methylguanine5-methyiainomethyluracii,5-methoxyaminomthy1--hourit beta D-inannosylqueosine, 5'-nethoxvcarboxvmethyluracil, 5-inethoxvuracil, 2-methylthio-N6 isopentenyladenine, uracil-S oxyacetic acid (v), wybutoxosine, pseudouracil, queosine. 2-thiocytosinc, 5-methyl-2thiuracil,2-rhiouracil.4-thiOuracil, 5-mehyluracil uracil-5 oxyacetic acid merthylester, uracil-5-oxyaceuic acid (v),5-mthyl-2hiourciI,3-3-amino 3-N-2-carboxypropyl uracil, (acp3)w, and 2,6-d.iaminopurine. Small nucleic acids and/or antisense oligonucleotides may also comprise at least one modified sugar moiety selected from thegroup including butnotlimitedtoarabinose, 2-fluoroarabinose, xvlulose, and hexose. In certain embodiments, a compound comprisesan oligonucleotide (e.g, a miRNA or miRNA encoding oligonucleotide) conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide.In certain such enbodiments, the moiety isa cholesterol moiety (e.g, antagomirs) ora lipid moiety or liposome conjugate. Additional moietiesfor conugation include carbohydrates, phospholipids, biotin, phenazine, fiblate, phenanthridineanthraquinone,acridine, fluoresceinsrhodamines, coumarins, and dyes. In certain embodiments, a con jugate group is attached directly to the oligonucleotide, In certain embodiments, a conjugate group is attached to the oligonucleotide by a linking moiety selectedfrom amino, hydroxyl, carboxylicacid, thiol unsaturations (e.g, double or triple bonds). 8-amino-3,6 dioxactanoic acid (ADO), succinimidyl 4(Nmlciidomethy)cyclohexane-1 carboxylate (SMCC), 6-aminohexanoicacid (AIEX or AHA) substituted C-C1 alkyL

-T3- substituted or unsubstituted C2-C0 alkenyl,and substituted or unsubstituted C2-C10 alkynyl In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, ntro, thiol, thloalkoxy halogen, alkyl, aryl, alkenyl and alkyny In certain such embodiments, the compound comprises the oligonucleoide having one ormre stabilizing groups that are attached to one or both termini of the oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell The cap can be present at the 5terminus(5Dcap), or at the 3'terminus (3 cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps. Suitable cap structures include a 4%5-methylene nucleotide, a I-(beta-D erythrofturanosyl) nuceotide, a 4thio nucleotide, a carbocyclic nucleotide, a 1,5 anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 34'seco nucleotide, an acyclic 3,4-dihydroxybutylnaucleitide, an acyclic 3,5 dihydroxypentyl nucleotide, a 3'Mverted nucleotide moiety,a 3'inverted abasic moiety, a 3-Tinverted nucleoide moiety, a 3-'-inverted abasic moitcy, a 1,4-butanediol phosphat 3phosphoramidate, a hexylphosphate, an aminohexyl phosphate,a 3 phosphae, a phsphorothioa a phosphorodihioate abridging ensthylphosphonate moiety, and a non-briding methylphosphonate moiety Samino.-alkyl phosphate, a 1,3 diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2 aminododecyl phosphate, a hydroxypropyl phosphate, a 5`5inverted nucleotide moiety, a 5'5'invertedabasicmoiety, a 5'phosphoramidate, a 5'~phosphorothioate, a 5-amino, a bridging and/or non-bridging 5-phosphoramidate, a phosphorothioate, and a 5'-nercapto maoiety. Small nucleic acids and/or antisense oligonucleotides can also contain a neutral peptide-like backbone. Such moleculesare termed peptide nucleic acid (NA)-oligomers and are described, e.g, in Perrv-O'Keefe et at (1996) Proc.Natt Acad. Sci. U.S.A. 93:14670 and in Eglom et at (1993) Nature 365:566. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, small nucleic acids andorantisenseoligonucleotides comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof In a further embodient, small nucleic acids and/or aintsense oligonucleotides are a.-anomerie oligonucleotides, An a-anomerie oligonuceotide forms specific double stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaimer et i X(1987) Nucl. Acids ResI 15:6625-6641). The olhgonucleotide is a 2' 0-methylribonuclootide (Inouc et al (1987) Nuc Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoeet aL (1987) FEBS Lett, 215:327 -330). Smallnucleic acids and/or antisense oligonucleotides of the methods and compositions presented herein may be synthesized by standard. methods known in the art, e. by use ofan automated DNA synthesizer (such asare commercially available from Biosearch.Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stei e al (1988) Nucl Acids Res, 16:3209, methylphosphonate oligonucleotides canbe prepared byuse of controlled pre glass polymer supports (Sarin e a (1988ProcNatAcdSci. USA. 85:74487451), etc. For example, an isolated miRNA can be chemically synthesized or recombinantly produced usingmethodsknownintheart. In some instances, miRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramoidites and a conventional DNA/RNA synthesizer. Commercialsuppliers of synthetic RNA molecules or synthesis reagents include,e. Prolig o(Hamburg, Germany), Dhariacon Research (Lafayette, Colo., USA'), Pierce Chmical (part of Perbio Science, Rockford, I., USA), Glen Research (Sterling Va., USA), ChemGenes (Ashland. Mass, USA), Cruachem (Glasgow, UK), and Exiqon (Vedback, Denmark) Small nucleic acids and/or antisense oligonucleotides can be delivered to cells in vivo_ A number ofmethods have been developed for delivering small nucleic acids and/or antisense oligonucleotides DNA or RNA to cells; eg., antisense molecules can be injected directly intothe tissuesite, or modified antisense molecules, designed to target the desired cells (e.g, antisense linked to peptidesor antibodies that specifically bid receptors or antigens expressed on the target cell surface) can be administered systematically.

In one embodiment. small nucleic acids and/or antisense oligonucleotides may comprise or be generated from double stranded small interfering RNAs (siRNAs), in which sequences fully complementarto cellular nucleic acids (e.g nIRNAs) sequences mediate degradation or in which sequences incompletely complementary to cellularnucleic acids (e,g, niRNAs) mediate translational repression when expressed within cells. In another embodiment, double stranded siRNAs can be processed into single stranded antisense RNAs that bind single stranded cellular RNAs (eg. microRNAs) andinhibit their expression. RNA interference (RNAi)is the process ofsequence-specificpost transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced genc, in vivo, long dsRNA is cleaved by ribonuclease Ul togenerate 21- and 22-nueleotide siRNAs. It has been shown that 21-nucleotide siR\A duplexes specifically suppress expression ofendogenous and heterologousgenes in differentmnniancel lines, including human embryonic kidney (293)andHeLa cells (Elbashireta(001)Nature411:494498) Accordingly,translation of a gene in a cell can beinhibited by contacting- the cell with short double stranded RNAs having a length of about 15 to 30 nuclotides or of about 18 to 21 nucleotides or of about 19 to 21 nucleotides. Alternatively, a vector encoding for such siRNAs or short hairpin RNAs (shRNAs) that are mtabolized into siRNAs can be introduced into a target cell (see, e.g.MciManus et al (2002) RNA 8:842; Xia el al (2002) Nature Biotechnology 20:1006 and Brummelkamp eclal (2002) Science 296:550). Vectors that can be used are conunercialyavailablec g., fror OligoEngine under ihe nume pSuper RNAi Systemr Ribozymt molecules designed to catalytically cleave cellular mRNA transcripts can also be used to prevent translation of cellular mRNAs and expression of cellular polypeptides, or both (See, e.g, PCTintcrnational Publication W090/l1364, published October 4, 1990; Sarver ecaT (1990) Science 247:1222- 1225 and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNAat site specific recognition sequences can be used to destroy cellular mRNAs, the use of hammerhead ribozymes is preferred Hammerhead ribozyres cleave rnRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5UG-3 The construction and

production of hammerhead ribozynes is well knownin the art and is described morefully in Hseloff and Gerlach (1988)Naure 334:585-591.The ribozymemayheengineered so thatthcleavagerecognionsite is locatednearthe5' end ofcellarmRNAs; i,e, to increase efficiency and minimize the intracellularaccumulationof non-functional mRNA transcripts. The ribozymes of the methods and compositions presented herein also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") suchas the onewhich occurs naturally in Tetrahymen thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thornas Cech and collaborators (Zaugl et al. (1984) Science 224:574-578; Zamu, at (1986) Science 231:470475;Zaug, et al (1986) Nature 324:429~433; published international patentapplication No. WO88/04300 by University Patents i.; Been, et al (1986) Cell 47:207-216). The Cech-type ribozyrnes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The methods and compositions presented herein encompasses those Cech-type ribozynmes which target eight base-pair active site sequences thatare present in cellular genes. As in the antisense approach, the ribozymes can be composed of modified ohgonucleotides (e g.for improved stability, targeting etc-). A preferred method of deliveryinvolves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol1III or pol 11 promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous cellular messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription of cellular genes are preferably single stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex. Nucleotide sequences may be pyrimidine-based which will result in TAT and CGC triplets across the threeassociated strands ofthe resulting triple helix, The pyrimidine-rich molecules provide base complementarity to a purine-rich region ofa single strand ofthe duplex in a parallel irientation to thatstrand. In addition, nucleic acid molecules hay be chosen that are purine-rich, for example, containing a stretch of i residues. These molecules will form a triple helix witha DNA duplex that's rich in GC pairs, in which the majority ofthepurine residues are located on asinle strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback moleculesare synthesized in an alternating, 5 5'manner, such that they base pair with first one strand of a duplex and then the other, eliminating thenecessity fora sizable stretch of either purines or pyrimidines to be present on one strand of a duplex. Small nucleic acids (eg, miRNAs, pre-maiRNAs, pri-miRNAs, miRNA*, anti miRNA, or a miRNA binding site, ora variant thereof antisense oligonucleotides, ribozyms and triple heixmolecules of themethods and compositions presented herein may be prepared biy any tthod known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyibonucleotides and oligoribonuleotides well known inthe artsuch as for example solid phase phosphoramidite chemical synthesis. Altenatively, RNA molecules may be generated by invitro and in vvo transcription of DNA sequences encoding the antisenseRNAmoeculeSuch DNA sequences may beincorporatedintoa wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase proroters, Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively orinducibly, depending on the promoter used, can be introduced stably into cell lines. Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5'and/or 3' ends of the molecule orthe use of phosphorothioate or 2 0-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotidcbackbone, One of skill in the art will readily understand that polypeptides,small nucleic acids, and antisense oligonutleodes can be further linked to another peptide or polypeptide(ag a heterologous peptide), eg- that serves as a means of proteindetection. Non-limiting examples of label peptide or polypeptide moieties useful for detection in the invention include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase beta-galactosidase, r acetylcholinesterase; epitope tags, suchas FLAG, MYC, HA, or HIS tags; fluorophores such as green fluorescent protein; dyes; radioisotopes; digoxygeuin; biotin; antibodies; polymers; as wellas others know in the art, for example, in Principles of Fluorescnce Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999)

In addition to the agents described herein, additional agents are particularly useful for upregulating or downregulating Immune responses accordingtothepresentinvntion. For example, modulation of the interaction between PD- and PD- ligand (eg soluble PDL1) (ag,membrane-bound PD- I and/or soluble PD-1), or between PD-1 ligand (eg., soluble PD-Li ) (eg, membrane-bound PD-L Iand/or soluble PD- I) and a B7 polypeptide, results in modulation of the immune response. In general,in embodinnts where PD-Libinds to a costimulatory receptor such as B7 - ,upregulation of PD-LI activity results in upregulation of immune responses, whereas downregulation of PD-Li activity results in downregulation of immuneresponses. In embodiments where PD-L binds to inhibitory receptors such as PD-, upregulation of PD-L Iactivity results in downregulation of immune responses, whereas downreguation of PD-L. activity results in upregulation of immune responses. It isalso believed that soluble forms of PD-Li, whether naturally occurring or cleavage products of membrane-bound PD-L, can still interact with PD-L Ireceptors, such as B7- Ior PD-L1, to modulate immune responses as the membrane bound version. Non-initing examples of how such agents can modify immune responses include the observation rhar the interacion betweena B7 polypeptide and a PD- IIigand (eg soluble PD~LI) polypeptide prevents PD-1 ligand (e.g ,soluble PD-L I) from binding to PD-1 and, thus, inhibits delivery ofan inhibitory immune signal. Thus, in oneembodiment, agents which block the interaction between PD-i and PD-I ligand (eg, soluble PD-LI) can prevent inhibitory signaling- In one embodiment, agents that block the binding of a B7 polypeptide toa PD-1 ligand (eg. soluble PD-LI) polypeptideallowPD-1 igand(g, soluble PD-L1) to bind PD-1 and providean inhibitory signal to an immune cell PD-1 ligand (eg, soluble PD-1), by binding to a B7 polypeptide, also reduces the B7 polypeptide binding to the inhibitory receptor CTLA4. In one embodinentt. agnts that block the binding ofa B7 polypeptide to a PD- ligand (egsoluble PD-I.) polypeptide allow the B7 polypeptide to bind CTLA4,and provide an inhibitory signal to an immune cell.inanotherembodint.PD-LIbybindingtoaB7polypeptidealsoreducestheB7 polypeptidebindgtothecostimulatoryreceptorCD28Ths,inoneenbodiment,agents that block the binding of a B7 polypeptide to a PD-1 ligand (e.g,soluble PD-L1) polypepide allow the B7 polypeptide to bind CD28,and provide a costimulatory signal to an Imunne cell,

For example, in one embodiment, agents that increase the interaction between a PD 1 ligand (e.g, soluble PD-LI) anda B7 polypeptide can enhancean immune response while agents that decrease the interaction between a PD- ligand (e.g, soluble PD-I1) and a B7 polypeptide can reducean immune response by enhancing the interaction between the PD-1 ligand (eg, soluble PD~L) and PD-i and/ or the interaction between the B7 polypeptide andCTLA4. In one embodimentagents that modulate the interactionbetween a PD-1 ligand (e.g, soluble PD-L) and a B7 polypeptide do not produce inhibition of the interaction betweena PD-l ligand (e.g, soluble PD-L 1) and PD- Iand/ or between the B7 polypeptide and CTL A4, In another embodiment, agents that increase a PD-I ligand (e.g, soluble PD-Li) interaction with B7 polypeptide, also decrease the interaction between the PD-1 ligand (e.g, soluble PD-LI) and PDi, and/or between tie B7 polypeptide and CTLA4 In yet another embodiment, agents that decrease the interaction of a PD- ligand (eg, soluble PD-LI) and aB7 polypeptidenbance or increase the interaction between the PD-1 ligand (ag, soluble PD-LL) and PD-I, andorbetween the B7 polypeptide and CTLA4 Exemplary agents r mocdating (eg, reducing) an immune response include antibodies againstPD-1, a PD-1 ligand (eg..soluble PD-LI), or a B? polypeptide which inhibit the interaction of the PD-I ligand (eg, .soluble PD-1) with PD or B7 polypeptide; bispecific antibodies that enhance PD-1 signaling, suchas bispecific antibodies against PD-I and PDL; agents that reduce the expression of inhibitory receptor-ligand interactions, such as antisense nucleic acid molecules, triplex oligonueeotides, or ribozymes targeting PD1 and/or PD-II; smanl molecules or peptides which inhibit the interaction of the PD ligandg, soluble PDL1) with the B7 polypeptide; and fusion proteins (g, the extracellular portion of the PD1 ligand (e.g, soluble PD-J) or B7 polypeptide, fused to theFc portion of an antibody) which bind to the B7 polypeptide or PD land (g, solube PD-Li), respectively,and prevent the interaction between the PD-I ligand (eg, soluble PD-L1) and B7 polypeptide. In another enibodiment, agents that increase the interaction between a PD-1 ligand (eg, soluble PD-L) and aB7 polypeptide, decrease an iinmue response by decreasing the ability of the B7 polypeptide to bind to CD28. In yet another embodiment, agents that decrease the interaction between a PD-I ligand (g., soluble PD-L1) and a B7 polypeptide can increase the inrune response by increasing the interaction between the B7 poilypeptide and CD28.

Agents that modulate the interaction between a PD-l ligand (eg, soluble PD-UL) and a PD-I polvpeptide can also be used toup or down regulate the immune response. For example, agents that increase the interaction between the PD-1 ligand (e., soluble PD- 1) and PD-1 polypeptide can decrease an immune response while agents that decrease the interaction between the PD- ligand (e,g, soluble PD-LI) and PD-Ipoypeptide can increasennmmunePresponse. Pfierably, agents that modulate theinteraction between the PD-1 ligand (g , soluble PD-L)and PD-1, do not modulate (have no direct affect on) the interaction between the PD-l ligand (e.g, soluble PD-L1.) and a B7 polypeptide. Inanother embodiment, agets that increase the interaction between the PD-I ligand (eg.,soluble PD Li)and PD-, decrease the interaction between the PD-1 ligand (eg, soluble PD-Li)and the B7 polypeptide. In yetanother embodiment, agents that decrease the interaction between the PD-Li ligand and PD-1 increase the interaction between the PD-1 ligand(eg, soluble PD-LI) and the B7 polypeptide. Exemplary agentsFormodulating (eg. enhancing) an immune response include antibodies against PD-1 ora PD-1 ligand (.g, soluble PD-LI) which block the interaction between PD-] and the PD-1 ligand(eg, soluble P-LI); bispecific antibodies that enhance 87 signaling, suchas bispecificantibodies against PD-LI and 37- ; multivalent antibodies against such a target thatligate many suchmolecules together in order to increase local concentrations and stimulate intetions;agents that reduce the expression of costimulatory receptor-ligand interactions' such as antisense nucleic acid molecules, triplex oligonucleotides, or ribozymes targeing B7-1; small molecules or pepides wicch block theinteraction between PD-1 and the PD-I ligand (e.g, soluble PD-L1.);and fusion proteins (e g, the extracellular portion of a PD-I ligand (e.g, soluble PD-L) or PD-I polypeptide fused to the Fe portion of an antibody) which bind to PD-1 ora PD- I ligand (eg, soluble PD-L 1) and prevent the interaction between the PD ligand (e.g, soluble PD~L1) andPD- I In some embodiments, agentsuseful for upregulating or downregulating PD-1 and/or PD-L Iin particular are useful. Combinations of any such agents are contemplated, Exemplary agents for use in downmodulating PD-LI (i.e., PD-LI antaonists) include (for example): antisense molecules, antibodies that recognize PD-L1, coipounds that block interaction of PD-LI and one ofitsnaturally occurring receptors ona immune cell (e,g, soluble, monovalent PD-LI molecules,and soluble forms of137-4 ligands or compoundsidentified in the subject screeningassays). In some eibodiments, combinations of antibodies that target either the membrane-bound PD-L1 form or the soluble PD-LI formare useful for fnctionally inactivating both forms of PD-L Exemplary agentsfor use in downmodulating PD-I (I .e PD-] antagonists) include (for example) arntisense molecules, antibodies that bind to PD-1, bta do not transduce an inhibitory, signal to the immune cell C"non-activatng antibodies"), and soluble forms of PD 1 Exenplary agents for use in uprnodulating PD-LI (i.e., PD-L agonists) include (for example): nucleic acid molecules encoding PD-LI polypeptides, multivalent forms of PD L1, compounds that increase the expressionof PD-Liand cells that express PD-LI, and the like. Exemplary agents for use in upiodulating PD- (i.e., PD-i agonists) inchide (for example); antibodies that transmit an inhibitory signalviaPD-1,compoundsthatenhance the expression of PD-1, nucleic acid molecules encoding PD-I, andformsofB7-4that transducea signal via PD-1 The modulatory agents described herein (eg_ antibodies, small molecules, peptides, fusion proteins, or small nucleicacids) can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The compositions may contain single suchmoleculeoragentoranyconibinationofagentsdescribedhereinBasedonthe geneict. paihway analyses described herein, it is believed that such combinations ofagents is especially effective in diagnosing, prognosing, preventing, and treating cancer. Thus, "single active agents" described herein can be combined with other pharmacologically active compounds ("second active agents") known in the art according to the methods and compositions provided herein. It is believed, that certain combinations work syn-rgistically in the treatment of particular types of cancer. Second active agents can be large olecules (e.g., proteins) or small molecules (e.g. synthetic inorganic, organometallic, or organic molecules), Examples oflargemolecule active agents include, but are not limited to, hematopoictic growth factors, cytokines, and monoclonal and polyclonal antibodies. Typical large molecule active agents are biological molecules, such as naturally occurring or artificially made proteins. Proteins that areparticularly useful in this invention inchide proteins that stimulate the survival andir proliferation ofhenatopoietic precursor cells and imrmnologically active poitic cells in vilro or in vivo. Others stimulate the division and differentiationofcommitted crythroid progenitors incells in vitro or in vivo Particular proteins include, but are not limited to: interleukins, such as IL-2 (including recombinant IL-Il ("riL2") and canarypox IL-2), , IIL-12, and IL-1i8; interferons, such as interferon alfa-2a, interferon alfa-2b, interferon alpha-ni, interferon alpha-n3, interferon beta-la, and interferon gamma-lb; M-CF and GM-CSF; and EPO. Particular proteins that can be used in the methods and compositions provided herein include, but are not limited to: filgrastim, which is sold in theUnited States under the trade name Neupogen@ (Amgen, Thousand Oaks, Calif.); sargramostim, which is sold inr the United. States under the trade name Leukine(Immanex, Seattle, Wash.);and recombinant EPO, whichis soldin the United States under the trade name Epogen@ (Amgen, Thousand Oaks, Calif) Recombinantand mutated forms of GM-CSF can be prepared as described in U.S. Pat Nos, 5,391,485; 5,393,870; and 5,229,496; all of which are incorporated herein by reference, Recorbinantand mutated forms of G-CSF can be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291;5,528,823;and 5,580,755;all of which are incorporated herein by reference, Antibodies that can be used in combination form include monoclonal and polyclonal antibodies. Examples of antibodies include, but are not limited to, trastuzumab (Hlerceptin@), rituximab (Rituxan@), bevacizumab (Avastinv), pertuzumab (Omnitargt, tositumonab (Bexxar@), edrecolomab (Panorex:),and G250, Comiounds of the invention can also be combined with, or used in conination withani-TNF-a antibodies, Large moleculeactive agents may be administered in the form of anti-cancer vaccines. For example, vaccines that secrete, or cause the secretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used in the methods, pharmaceutical compositions, and kits provided herein. See, e L, Eens L. A, et al., Curr. Opinion Mo ,Ther, (1): 77-84 (2001), Second active agents that are small molecules can also be used to in combination as provided herein. Examples of small molecule second active agentsinclude, butare not limited to, anti-canceragents, antibiotics, immunosuppressive agents, and steroids, some embodiments, well known "combination chemotherpy" regimens can be used. In one embodiment, the combination chemotherapy comprisesa combination of two or more of cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin or adriamycim), ncovnrin (incristine), and prednisone, In another preferred embodiment, the combination chemoerapy coimprises a combination of cyclophsophamide, ecovorin, prednisone, and one or more chemotherapeutics selected from the group consisting of anhiracycline, hydroxydaunorubicin, epirubicin, andimotixantrone, Examples of other anti-cancer agents include, but are not limited to: acivicin; aclambicin; acodazole hydrochloride;aronine;adozelesin; aldesleukin; altretamine; ambonycin; ametantroneacetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomin; batimasat; benzodepa; bicalutamide; bisantrene hydrochloridei bsnafide dimesylate; bizelesin; bieomycin sulfate brequina sodium; bropirimine;busulfan; cactinorycmi;calusterone; caracemide; carbetimer, carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefigol; celecoib (COX-2 inhibitor); chiorambuci;ciroltemcin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine dactinomycin; daunorubicin hydrochloride; decitabine dexormaplatin; dezaguanine; dezaguanintmesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone popionate; duazomycint edatrexate; eflornithine hydrochloride; elsamitrucin;eloplatin; enpromate; epipropidine; epirubicin hydrochloridet erbulozole; esorubicin hydrochloride; estrimustine; estramustine phosphate sodium; ctanidzvole; etoposide; etoposide phosphate; eoprne; fadrozole hydrochiiride; fazarabiefenetinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecinsodium; gemitabine; gemitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irintecan; irnotecan hydrochloride; lanreotideacetate; letrozole; leuprofide acetate; harozole hydrochloride; lonetrexol sodiurn; lonustne; losoxantrone hydrochloride; masoprool r matnslnitne mechlorethamine hvdrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metopn meturedepa mitindomide; mitocarcin; mitocronin; mitogillin; mitomain; mtonycin mitsper; mnittatie;itoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormapatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycinsulfate; perfosffamide; pipobroman; piposulfin; piroxantrone hydrochlorideimytinploniestane; pormfi ersodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofrin; riboprine; safingol; safingol hydrochloride; semustine; simtrazene;sparfosate sodium; sparsomyci; spirogermanuam hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisonixn; tecogalan sodium; taxotere; tegafur; teloxanrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin tirapazamine; toremifene citrate trestolone acetate; triciribine phosphate; trinetrexait trimetrexate lucuronate;tiptorein; bulozole hydrochloride; uracil mustard. uredepavapreotide; verteprfin; vinblastine sulfate; vineristine sulfate; vindesine; vindesine sulfite; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorabicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone: acarubicin;acyfuvene;adecypenol: adozelesin; aldesleukin; ALL-TK antagonists; atretamine;ambamustine;amidox; arnifbstine; aminolevuinic acid; arubicin; amsacrine; anagrelide aLnastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist U;ntrelix;anti dorsalizing morphogenetic protein-1; antiandrogen, prostatic carclinoma; antiestrogen; antineoplaston antisense oligonuclcotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apuinic acid; ara-CDP-DL-PTBA; arginine deamninasc asuinee: atamiestane; atrimustinle axinainstatitaixinastain2xinastatin 3; azasetron; azatoxin; azatyrosine;baccatin III derivatives; balanol; batimastat; BCRABL antagonists benzochlonins;benzoylstaurospoini beta lactam derivatives; beta-alethine; btaclanyci B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; biaziridinylspermine; bisnafide; bistratene A: bizelesin; breflate; bropirimine; budotitane; buthionine sufoxinIne calcipotriol; calphosUn C; camptothecin derivatives; capecutabin; carboxamide-amino triazole; carboxyanidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetroreix; chlorins; chloroquinoxaline sulfonmide;cicaprost; cis-porphyrinn cladribine; clomifene analogues; clotrimazole; collisnycn A; collismycin B; combretastatin A4; combretastatin analogue;conagcain; crambescidin816; erisnatolt eryptophycin 8; crypophycin A derivatives; Curacin A; clopentanthraquinonescycloplatam; cyclosporinA; cypemycin; cytarahine ocfosfate; cytolytic factor; cytostatin; daciximab; decitabine; dehydrodidenini B; deslorelin dexamethasone dexifosfamide; dexazoxane; dexverapamil; diaziquone; dideinin B; didox; diethynorspermine;dihydro~5-azacytidine; ibdirotaxol, 9-; dioxamycin; diphenyl spiromustine; docaxel; docosano; dolasetron; doxifluridine; doxorubicin; droloxiene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfibsine: edrecolomab;eflornithine; e eneemitit;epirubicin; epristeride; estranustine analogue;estrogea onistsNtrogen antagnits etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol flezelastine fluasterone; fludarabine; fluorodaunorunicin hydrochlorde; forfenumcx; formestane; fostriccin; ftemustinL;gadoliniui texaphyrin; galliun nitrate; galocitabine; ganirelix gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifenc; idramantone; ilmofosine; ilomastat; imatinib (e,g Glcevc4), imiquimod; immunostiniulantpeptides; insulin-like growth factor-I receptor inhibitor interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole;isohomohalicondrin B;itasetron; jaspliakinolide; kahalalide F; lamellarin-N triacetate; lanreoride; leinamycin; lenograstim; leninan sulfate; leptolstatin; letrozole; leukemia inhibiting factor;leukocyto alpha interferon; leuproidet-estrogen-progesterone; IeULprorelin levamisole; liarozole; linearpolyamine analogue lipophilic disaccharide peptide; lipophlic platinum compounds;I issoclinamide 7; lobaplatin; ombricine; lonetrexol; lonidamine; losoxantrione loxoribine;atrtotecan; lutetium texaphyrin; lsofylie;lyticpeptides; maitansine; mannostatin.A imunastat; masoprocol; maspin; matrilysin inhibitors; matrix mualloprotcinase inhibitors;menogaril; tmerbarone; meterelin; metioninase;metolop ide MIFinhibitor; mifepistone; miltefosine; mirimostim; nitoguazone;n itolactol; mitomycin analogues; mitonafide; mitotoxinfibroblastgrowthfactor-saporin; mitoxantrone; mofarotene; mogramostim; Erbitux, human chorionic ganadotrophia; monophosphoryl ipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinalie; N-substituted benzaindes; niafaelin; nagrestip; naloxonc+pentazocine napavin;naphterpinnatorast nedaplatinnmorubcin; neridronic acid; nilutanide nisamycn;nitric oxide modulators nitroxide antioxidant; nitrullyn; oblimersen(Genasensc@); 06-benzylguanine; octrotide okiceonoe; oligonuclcotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclituxel analogues; paclitaxel derivatives; palauamine; palitoyirhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine;p egaspargase; peldesine;pntosan polysulfate sodium; pentostatin; pentrozole perflubron; perfosfamide; perillyl alcohol phenazinoinycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin;piritrexm; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinumcmpounds; platuinu-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibtrs; protein A-based imunne nodulatorr; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpirins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylen conijugate; raf antagonists; raltitrexed; ramoseiron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retellipiine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;sagramiostim; Sdi I mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibiors; sizofuran; sobuzoxane;sodium borocaptate;sodiumphnyleate; solverol; somatomedin binding protein; sonenmin; sparfosic acid; spicamcin D; spiromustine splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive asoactive intestinal peptide antagonist; suradista suramin; swainsonine; tallimustine; tanoxifen methiodide; tauromnustine;tazarotene; tecogalan sodium; tegafur, tellurapyryium; telomerase inhibitors; temoporfin; teniposide; tetrachlorodecaoxide;ltcrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoletin mimetic; thymalfasinthymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsntim; toremifene; translation inhibitors; tretinoin; triacetyluridine;trciribine; trimetrexate; triptorclir; tropisetron; urosteride; tyrosine kinase inhibits; tyrphostins; UBC inhibitrs; uberumex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists vapreotide; variolinB; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; andzinostatin stimalamer Specific second active agents include, but are notlimited to, chlorambucil, fludairabine, dexamethasone (Decadront' hydrocortisone, methyliprednisolone, cilostamide, doxorubicin (Doxicl), forskolin. rituximab. cyclosporin A, cisplatin, vincristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual PDE4/7 inhibitors such as IR-284, cilostazol, meribendan, mirinone, vesnarionone, enoximone and pimobendan, SVk inhibitors such as fostaniatinibdisodium (R406/R788), R343, R-112 and Excellairt (ZaBeCor Pharmaceuticals, Bala Cynwyd, Pa.).

Ilk.. Methods of Selectit Agents and Compositions Another aspect of the invention relates to methods of selecting agents (eg. antibodies, fusion proteins, peptides, small molecules, or smill nucleic acids) which bind. to, upregulate, dowuregulate, ormodulate one or more biomarkers of the invention listed in Table I and the Examples and/or a cancer (ega head, neck, orlung cancer), Such methods can use screening assays, including cell based and non-cell based assays.

In one embodiment. the invention relates to assays for screening candidate or test compounds which bind to or modulate the expression or activity level of, one or more biomarkers of theinvention, including one or more biomarkers listed in Table I and the Examples, or a fragment thereof Such compounds iclude, without limitation,antibodies, proteins, fusion proteins, nucleic acid molecules, and small molecules, Inone embodimnt, anassay is a cell-based assay, comprising contacting a cell expressing one or morc bionarkers of the invention, including one or more biomarkers listed in Table 1 and the Eximples, or a fragment thereof, with a test compound and determining the ability of the test compound to modulate (e.g, stimulate or inhibit) the level of interaction between the biomiarker and its natural binding partners as measured by direct binding or by measuring a parameter of cancer. For example, in a direct binding assay, the biomarker polypeptide, a binding partner polypeptide of the biomarker, or a fragment(s) thereof, can be coupled with a radioisotope or enzymatic label snch that bindg of the biomarker polypeptide or a fragment thereof to its natural binding partners) or a fragment(s) thereof can be determined by detecting the labeled molecule in a complex, For example, the biomarker polypeptide, a binding partner polypeptide of the biomarker, or a(frament(s)thereofcan be labeled with''"S, "Cor H, either directly or indirectly, and the radioisotope detected by direct Counting of radioemiission or by scintillation counting. Alternatively, the polypeptides of interest a can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatc label detected by detenirinaion of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine theability of a compound to modulate the interactions between one or more biomarkers of the invention, including one or more bioiarkers listed in Table I and the Examples, ora fragment thereof, and its natural binding partner(s) or a fragment(s) thereof.without the labeling of any of the interactants(e.g. using a microphysiometer as described in McConnuell, H. M. el (1992) Science 257:1906-1912), As used herein, a"microphysiometer (e.g. Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sesor (LAPS). Changes in this acidification rate can be used as an indicator of theinteraction between compound and receptor. In a preferred.embodiment, determining the ability of the blocking agents(e.g, antibodies, fusion proteins, peptides, nucleic acid molecules, or small molecules) to antagonize the interaction between a given set of poYpeptides can be accomplished by determining the activity of one or more members of the set of interacting molecules. For example, the activity of one or more biomarkers of theinvention, dilung one ormore biomarkers listed inTable I and the Examples, ora fragment thereof, can be determined by detecting induction of cytokine or chemokine response, detecting catalyticenzymatic activity of an appropriate substrate, detecting the induction ofa reporter gene (cmprisinga target-responsiveregulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g, chloramphenicol acetyl trinsferase), or detectinga cellular response regulated by the bioiarker or a fragment thereof (g, modulations of biological pathways identified herein, such as modulated proliferation, apoptosis, cell cycle, and/or ligand receptor binding activity) Determining the ability of the blocking agent to bind to or interact with said polypeptide can be accomplished by measuring theability of an agent to modulate immune responses, for example, by detecting changes in typeand amount of cytokine secretion, changes in apoptosis or proliferation, changes in gene expression or 1.5 activity associated with cellular identity, or by interfering with the ability of said polypeptide to bind to antibodies that recognize a portion thereof. In yet another embodiment, an assay of the presentinveuntion is a cell-free assay in which one or more biomarkers of the mention,including one ormore biomarkers listed in Table I and the Examples or a fragment thereof, eg, a i activefagment thereof, is contacted with a testcompound, and the ability of the test compoundtobindtothe polypeptide, or biologically active portion thereof, is determined . Bindingofthe test compound to the biomarker or a fragment thereof, can be determined either directly or indirectly as described above. Determining the ability of the biomarker or a fragment thereof to bind to its natural bindingpartners) oraaents)thereofcanalso be accomplished using a technology such as atimeBiomolcularInteractionAnalysis (BIA) (Sjolnder, S. and Urbaniczky, C. (1991)Anal Chen 63:2338-2345and Szabo eta. (995)Crr. OpinStruct, Biol 5:699-705). As used heroin, "BIA" is a technology for studying biospecific iteractiols in real time, without labelingany of the interactants (eg, BIAcco), Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biologicalpolypeptides.Oneornure biomarkers polypeptide or a fragment thereof can be immobilized on a BlAcore chip and multiple agents, eg, blocking antibodies, fusion proteins peptides, or small molecules can be tested for binding to the immobilized biomarker polypeptide or fragment thereof

An example of using the BIA technology is describedby Fitz e al. (1997) Oncogene 15:613, The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins. In the case of cell-freeassays in which a membrane-bound form protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound fori of the protein is maintained insolution, Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n dodecylglucoside, n-dodecylaltoside, octnoyl-Nimethlglutcamid, decinoyl-N

methylglucamide, Tritonk X-100, Triton X-114, Thesit Isotridecypoly(ethylene glycol ether). 3{(3eholamnidopropyl)dimethlamminio--propane suifonate (CHAPS). 3~[(3

cholamidopropyI)dimethylamminio]-2-hydroxy--propane sulfonate (CH- APSO), or.N dodecyl=N,N-dimethyl-3-amonio-1-propa sulforate, In one or more embodiments of the above described assay methods, it may be desirable to immobilize either the biomarker polypeptide, the natural binding partner(s) polypeptide of the biomarker, or fragments thereof, to facilitate separation of complexed fromncomplexed.formsof oe or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound in the assay can be accomplished in any vessel suitable for containing theractants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, luhione-S-transferase-basefusion proteins, can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound, and the mixture meubated under conditions conducive to complex formation (eg, at physiological conditions for salt and pHl) Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example., as described above. Alternatively, the complexes can be dissociated from the matrix,and the levelof binding or activity determined using standard techniques, In n alternative embodiment, determining the ability of the test compound to modulate the activity of one or more biomarkers of the invention, including one or more biomarkers listed in TableI and the Examples, ora fragment thereof, or of natural binding partners) thereof can be accomplished by dererming the ability of the test compound to modulate the expression or activity of a gene, e.g, nucleic acid, or gene product, e.g, polypeptide, that functions downstream of the interaction, For example,inflammation (e.g, cytokine and chemokine) responses can be determined, the activity of theinteractor polypeptide on anappropriate target can be determined, or the binding of the interactor to anappropriate target can be determined as previously described. In another embodiment,modulators of one or more biomarkers oftheinvention including one or more biomarkers listed inTable I and the Examples, or a fragment thereof, are identified in a method wherein a cell is contacted witha candidate compound and the expression or activity level of the biomarker is determined, The level of expression of bionarker mRNA or polypeptide or fragments thereof in the presence of the candidate compound is compared to the level of expression of biomarker mRNA or polypeptide or fragments thereof in the absence of the candidate compound. The candidate compound can ihen be identified as a modulator of biomarker expression based on this comparison. For example, when expression of biomarker mRNA or polypeptide or fragments thereof is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimlator of bioarker expression. Alternatively, whenexpressionof biomarker mRNA or polypeptide or fragments thereof is reduced (statistically significantly less) in the presence of the candidate compound thanin its absence, the candidate compound is identified as an inhibitor of biomarker expression, The expression level of biomarker mRNA or polypeptide or fragmentsthereofinthecells can be detrmined by methods described herein for detecting bioimarker mRNA or polypeptide orfragments thereof. In yetanother aspect of the invention, a biomarker of the invention, including one or more biomarkers listed in Table I and the Examples, or a fragment thereof, can be used as "bait proteins" in a two-hybridassay or three-hybridassay (see, e g, U.. Pat.N'o. 5,283,31r7; Zeros et al (1993) Cell 72:223-232; Madura e al (1993) Biol. Chem 268:12046-12054; Bartel eta (1993) Biotechniques 14:920-924; iwabuchi et al (1993) Oncogene 8:16931696; and Brent W094/10300), to identify other polypeptides which bind to or interact with the biomirker or fragnients thereof amd are involved in activity of the biomarkers. Such biomarker-binding proteins are also likely to be involved in the propagaion of siguals by the bionarker polypeptides or biomarkernatural binding partners) as, for example, downstream elements of one ormore bionarkers -mediated signaling pathway.

The two-hybrid system is based on tie modular nature of mosttranscription factors, which consist of separable DNA-binding and activation domains. Briefly, the assayutilizes two different DNA constructs. In one construct, the gene that codes for one or more biomarkers polypeptide is fused toagene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4. In the other construt a DNA sequence, from a library oFDNA sequences, that encodes an unidentified polypeptide ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" polypeptides are able to interact in vivo, forming one or more biomarkers -dependent complex, theDNA-binding and activation domains of the transcriptionfactorarebroughtinto close proximity. This proximity allows transcription of a reporter gene (e.g, LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with one or more biomarkers polypeptide of the invention, including one or more biomarkers listed in Table I and the Examples ora fragment thereof In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell based or a cell-free assay, and the ability oftheagent to modulate the activity of one or more biomarkers polypeptide or a fragment thereof can be confirmed in vivo, eg, in an annual such as an anialmodel for eclular transformationand/ortaorigenesis This invention further pertains to novel agents identified by the above-described screeningassays, Accordingly, it is within the scope of this invention to further use an agent identified as described herein inan appropriate animal model. For example, anagent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, anagent identified as described herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein,

ll Uses and Methods of the Invention The bionarkers of the invention described hereinincluding the biomarkers listed in Table I and the Examples or fragments thereof, can be used in one or more of the following methods: a) screeningassays; b) predictive medicine (e.g, diagnostic assays, prognostic assays, and monitoring of clinical trials); and c)methods of treatment (eg, therapeutic and prophylactic, e.g, by up- or down-modulating the copy number,levelofexpressionand/or level ofactivity of the one or more biomarkers). The biomarkers described herein or agents that modulate theexpressionand/or activity ofsuch biomarkers canbe used, for example, to (a) express one ormore biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples or a fragmentthero(e.g1viaarecombinant expression vector in a host cell in gene therapy applications orsthetic nucleic acid molecule), (b) detect biomarker mRNA or a fragment thereof (eg.iIn a biological sample) or a genetic alteration in one or more bioniarkers gene, and/or (c) modulate biomarker activity, as described further below. Thebiomarkers or modulatory agents thereof can be used to treat conditions or disorders characterized by insufficient or excessive production of one or more biomarkers polypeptide or fragment thereof or production of biomarker polypeptide inhibitors- In addition, the biomarker polypeptides or fragments thereof can be used to screen for naturally occurring biomarker binding partner(s), to screen for drugs or compounds whichmodulate biomarker activity, as well as to treat conditions or disorders characterized byinsuficient or excessive production of biomarker polypeptide or a fragment thereof or production of bionmarker polypeptide forms which have decreased,aberrant or unwanted activity compared to biomarker wild type polypeptides or framents thereof (egcancers, including head, neck, and/orlung cancers).

A. Screening Assays In one aspect, the present invention relates to a method for preventing in a subject, a disease or condition associated with an unwanted, more than desirable, or less than desirable, expressionand/or activity of one or more biomarkers described herein. Subjects at risk for a disease thatwould benefit fiom treatment with the claimed agents ormethods can be identified, for example, by any one or combination of diagnostic or prognostic assays known in the art and described herein (see, for example, agents and assays described in Ifl. Methods of Selecting Auents and Cimpositions)

B.Predictive Medicine The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring of clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determiing the expression and/or activity level of biomarkers of theinvention, including biomarkers listed in Table I and the Exarples or fragments thereof in the context ofa biological sample (eg, blood, serum, celLs, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated withaberrant or unwanted biomarker expression or activity. The present invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with biomarker polypeptide, nucleic acid expression or activity. For example, mtrations in one or more bionarkers gencan be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to tien nset of a disorder characterized by or associated with biomarker polypeptide, nucleic acid expression or activitY, Another aspect of the invention perains to monitoring the influence of agents (cg., drugs, compounds, and small nucleic acid-based Inolecues) on the expression or activity of bionarkers of the invention, including biomarkers lised in Table I andr the Examplesorhfagm3ents thereof, in clinical trials.iThese and other agents arc described in further detail in the following sections.

1 Dinnostic Assays The present invention provides, in part, methods,systems, and code for accurately classifyingwhether a biological sample is associated with a cancer or a clinical subtype thereof (cg. head, neckand/or hung cancers). In some embodiments the present invention 2$ is useful for classifying a sample( front a subject) as a cancer sample using a statistical algorithm and/or empirical data , the presence or level of one or biomarkers described herein). An exemplary method for detecting the level of expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed inTableI1 and the Examples or fragments thereof, and thus useful fir classifying whether a sample is associated with cancer or a clinical subtype thereof (g head, neck, and/or lung cancers), involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of deecting the biomarker (eg, polypeptide or nucleic acid that encodes the biomarker or fragments thereof) such that the level of expression or activity of the biomarker is detected in the biological sample. in some embodiments, the presence or level of at least one, two, three, four, five, six, seven,eight, nine, ten, fifty, hundrc, or more biomarkers of the invention are determinedin the individual's sample in certain instances, the statistical algorithmis a single learning statistical classifier system Exemplary statistical analyses are presented in the Examples and can be used in certain embodiments, In other embodiments, a single learning statistical classifier system can be used to classify a sample as a cancer sample, a cancer subtype sample, ora non-cancer sample based upon a predictionor probability valueand the presence or level of one or more biomarkers described herein. The use of a single learning statistical classifier systeni typically classifies the simple as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/oroverall 8 accurac of at least about 75%, 76%,'77 , 78%, 79%80%, 82%83%,84%, 85%, 86%,87%88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%", or 99%. Other suitable statisticalallorithms are well knoviito those of skill in theart. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (.,panel of markers of interest) and making decisiOns based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g random forest) misused. I other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more leaning statistical classifier systems are used, preferablyintandem.Exampolearning statistical classifier systems include, but are not limited to, those using inducive meaning (e.g, decision/classificationtrees such as randomforests, Classification and regression tes (C&RT), boosted trees, etc .), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons suchas multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian leaning in belief networks, etc), reinforcement learning (eg,. passive learning ina known environment such as naive leading, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in anunknown environment. learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programing. Other leaning statisticalclassifier systems include support vector machines (e., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learn vector quantization (LVQ). In certain embodiments, the method of the present invention further comprises sending the cancer classification results to a clinician, e-g. an oncologist or hematologist. In another embodiment, the method of the present invention further provides a diagnosis in the form of a probability that the individual has a cancer or a clinical subtype thereo£ For example, theindividual canhave about a0%,5 10%, 15%, 21%25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater probabilityofhaxingcanceroraclinicalsubtype thereot Inyetanother embodiment, the method of the present invention further provides a prognosis of cancer in the individual, For example, the prognosis can be surgery, development of a clinical subtype of the cancer (eg, subtype of leukemia), development of one or more symptoms, development of malignant cancer, or recovery from the disease in some instances, the method of classifying a sample as a cancer sample is further based on the symptoms (eg, clinical factors) of the individual from which the sample is obtained. The symptoms or group of symptoms can before example, those associated with the IP. In some embodiments, the diagnosisofanindividualashavigcanceroraclinicalsubtypethereofistfbllowedby administering totheindividualatherapeuticallyeffetiveamountofadrug usefulfbr treating one or moresymptoms associated with cancer or the cancer. In some embodiments, an agent for detecting biomarker mRNA, genomic DNA, or fragments thereof is i labeled nucleic acid probe capable of hybridizing to biomarker nmRNA, genomic DNA, or fiagments thereof, The nucleic acid. probe can be, forexample, full-length biomarker nuclelicacid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions well known to a skilled artisan to biomarker mRNA or genomieDNA. Other suitable probesfor use in the diagnostic assays of the invention are described herein. In some embodiments, the nucleic acid probe is designed to detect transcript variants (i.e, different splice forms) of a gene. A preferred agent for detecting one or more biomarkers listed in Table I and the Examples or a fragment thereof is anantibody capable of binding to the biomnarker, preferably an antibody with I detectable label. Antibodies can be polvlonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.gn Fab or F(ab')2) can be used. The term"labeled", with regard. to the probe or antibody, Is intended to encompass direct labeling of the probe or antibody by coupling (e physically linking) a detectable substance to the probe or antibody, as wellas indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibodyand end-labeling ofaDNA probe with biotin sucI th'It it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within subject.That is, the detection method of the invention can be used to detect biomarker mRNA, polypeptidegenomic DNA, ortfragments thereof, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of bionar-ker mRNA or a fraginent thereofinclude Northern hybridizations and insitu hybridizations. In vitro techniques for detection of biomarker polypeptide include enzyme linked immunosorbentassays (ELISAs), Western blots, immiunoprecipitations and immnunofluorescence.in viro techniques for detection of biomarker genomic DNA or a fragfvment thereof include Southern hybridizations. Furthermore, in vivo techniques for detection of one or more biomarkers polypeptide ora fragment thereof include troducing into a subjectalabeled anti-bioiarker antibody, For example, the autibody cau be labeled with a radioactive marker whose presence and location iu a subject cau be detected by standard imaging techniques. In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain mRNA moleculesfrom the test subject or genomic DNA molecules from the test subject A preferred biological sample is a hematological tissue (eg, a sample comprising blood, plasma B cell, bone marrow, etc.) sample isolated by conventional means from subject. In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting thecontrol sample with a compound or agent capable of detecting polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof of one or more biomarkers listed in Table I and the Examples such that the presence of biomarker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of biomarker polypeptide, mRNA, cDNA, small RNAs,mature miRNA, pre-niRNA, pri miRNA, miRNA*, anti-miRNA, or a niRNA binding site, or a variant thereof, genomic DNA, or fragments thereof in the control sample with the presence of biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-niRNA, pri-miRNA, miRNA*,anti-miRNA, or a miRNA finding site, ora variant thereof, genomic DNA, or fragments thereof in the test sample. The inventionalso encompasses kits for detecting, the presence ofa polypeptide, mRNA, eDNA, small RNAs, mature miRNA, pe-miRNApri-miRNA, miRNA*,anti miRNA, or a miRNA binding-site, oravariant thereof, genomic DNA, or fragments thereof, of one or more biomarkers listed inTable 1 and the Examples ina biological sample. For example, the kit can comprise a labeled compound oragent capable of detecting one or more biomarkers polypeptide, mRNA, cDNA., small RNAs, mature miRNA, pre-iiRNA, pri-imiRNA, miRNA*,anti-miRNA, or a miNA binding site, ora variant thereof, genomic DNA, or fragments thereof in a biological sample;means for determining the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, niRNA*, anti-niRNA, or a miRNA binding site, or a variant thereof, genomic DNA. or fragments thereoff in the sample; and means for comparing the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-niRNA, pri-mriRNA, tiRNA*, anti-niRNA, or a miRNA bindinsite, ora variant thereof, genomic DNA, or fragments thereof, in the sample with a standard. The compound or agent can ie packaged in a suitable container. The kit can further comprise instructions for using the kit to detect the biomarker polypeLptide, mRNA, cDNA., small RNAs.mature miRNA, pre-miRNA, pri-miRNA, miRNA ani-miRNA,oramiRNA binding site, or a Variant thereof, genoric DNA, or fragments thereof, In some embodiments, therapies tailored to treat stratified patient populations based on the described diagnostic assaysare furtheradministered. 2 Prognostic Assays )5 The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression oractivity of one or more biomarkers of the invention, including one or more biomarkers listed inTable l and the Examples, or a fragment thereof As used herein, the tern "aberrant" inchides bionarker expressionor activity levels which deviates from the normal expression or activity In acontrol The assays described herein, such as the preceding diagnostic assaysor the following assays, can be utilized to identify a subject having orat risk of developing a disorder associated with a misregulation of biomarker activity or expression, such as ina cancer (e.ghead, neck, and/or lung cancers). Alternatively, the prognosticassays can be used to identify a subject having orat risk for developing a disorder associated with a misregulation of biomarker activity or expression. Thus, the present inventionprovidesa method for identifying and/or classifying a diseaseassociated withaberrant expressionor activity of one or more biomarkers of theinvention, including one or more biomarkers listed in Table1 and the Examples, or a fragment thereof, Furthermore, the progniostic assays described herein can be used to determine whethera subject can be administered an agent (e.g, an agonistantagonist, peptidomimetic, polypeptide, peptide,nucleicacid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant biotarker expression or activity. For example,such methods can be used to determine whether a subject can be effectively treated with an agent for a cancer (e.g., head, neck, and/or lung cancers). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant biomarker expression or activity hihatestsampleisobtainedand biomarker polypeptide or nucleic acid expression or activity is detected (e.g, wherein a significant increase or decrease in biomarker polypeptde or nucleic acid expression oractivity relative to a control is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant biomarker expression or activity). Insome embodiments., significant increase or decrease in biomarker expression or activity comprisesat least 2 2.1. 2.2, 23, 24, 25, 26, 27, 2,8, 29, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 775, 8, 8.5, 9, 9.5, 10, 10,5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 tines or roorehIigher oil er respectively,tha the expression activity or level of the marker ina control sample. The methods of the intention can also be used to detect genetic alterations in one or more bionarkers of the invention, including one or more biomarkers listed inTable 1 and the Examples or a fragment thereof, thereby determiningifa subject with thealtered biomarker is at risk for cancer (e.g, head, neck, and/or lung cancers) characterized by aberrant biomarkeractivity or expression levels. In preferred embodimenis, the methods include detecting, in a sample of cells front the subject, the presence or absence of a genetic alteration characterized by at least one alterationaffecting the integrity of a gene encoding one or more biomarkers polypeptide, or the.mis-expression of the biomarker (e.g, mutautons and/or splice variants). For example, suchgeneicalterationscan be detected by ascertaining the existence of at least one of 1) a deletion of e or more nucleotides from one or more biomarkers gene, 2) an addition of one or more nucleotides to one or more

_99- biomarkers gene, 3) a substitution of one or more nucleotides of one or more biomarkers gene, 4) a chromosomal rearrangement of one or more biomarkers gene, 5) an alteration in the level of a messenger RNA transcript ofone oroeiomarkers gene, 6) aberrant modification of one or more biomarkers gene, such as of themethylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of anessenger RNA iranscripi oFone or more biornarkers gene, 8) anonviid type level oF one ormore biomarkers polypeptide, 9) alelic loss of one or more biomarkers gene, and 10) inappropriate post-translational modification of one or more biomarkers polypeptide. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in one or more biomarkers gene. A preferred biological sample is a tissue or serum sample isolated by conventional meansfrom subject. In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Paienis 4,683,195 and. 4,683,202), such as anchor PCR or RACE PCR, or, altenatively, in aligation chain reaction (LCR) (see, ecg. Landegran et al (1988) Science 241:1077-1080;,and Nakazawa ct al (1994) Proc. NaiL Acad, Sci, USA 91:360-364), the latter of which can be particularly usefuldfor detecting poitinutatios in one or more biomarkers gene (see Abravaya et ad (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collectinga sample of cells from a subject, isolating nucleic acid (e.g, genomic DNA, mRNA, cDNA, small RNA, mature miRNA, pre-miRNA, pri-miRNA, rniRNA* anti-miRNA, or a miRNA binding site, or avariantthreof) from the cells of the sample contacing the nucleic acid sample with one or more primers which specifically hybridie to one ormore bioniarkers gene of the invention, including the biomarker genes listed in Table I and the Examples, or fragments thereof, under conditions such that hybridization and amplification of the biomarker gene (if present) occurs,and detecting the presence orabsence of an anplification product,or detecting thesize of the amplification productand comparing the length to a control sample, It isanticipated that PCR and/or LCR may be desirable to use as a preliminary aiplification step in conjunction with any of the techniques used for detecting nutations described herein. Alterative amplification methods include:self-sustained sequence replication (Guatelli J C. C af (1990) Proc, Nail Acad. Sci USA 87:1874-1878), trascriptiynal amplificationsystem(Kwoh, D, Y- et a/ (1989) Proc. Natl. Acad. Sci USA 86:1173-1177) Q-Beta Replicase (Lizardi, P. M. et al (1988) Bio-Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemesare especially useful for the detection of nuclic acid molecules if such molecules are present in very low numbers. In an alternative embodiment, mutations in one or more biomarkers gene of the invenon, including ne or more biomarkers listed inTable 1 and the Examples, or a fragment thereof, from a sample cell can beidetified by alterations in restriction enzyme cleavagepatterns. Forexample,sampleandcontrolDNA isisolatedamplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel lectrophoresis and compared, Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA Moreover, the use of sequence specific ribozyms (see, for example, US. Patent 5,498,531) can be used to score for the presence ofspecific mutations by developnIit or lossof a ribozyme cleavage site. In other embodiments, genetic mutations in one or more biomarkers gene of the invention, includingagene listed in Table 1 and the Examples, ora fragment thereof, can be identified by hybridizing a sample and control nucleic acids, eg, DNA RNA, mRNA., snall RNA, cDNA, mature miRNA.,premiRNA, pri-miRNA,.miRNA* anti-miRNA, or a miIRNA binding site, or a variant thereof, to high density arrays containinghundreds or thousands of oligonucleotide probes (Cronin, M. T. eia (1996) Hum. Muta7:244 55 Kozal, M,. et al (1996) Nat Med. 2:753-759). For example, genetic mutations in one or more biomarkers can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin e aL (1996) supra. Briefly, a first hybridization array of probes can be used to scan through long ttc ofDNAinasmpe dcontrto identify base changes between the sequences by making linearrays of sequential, overlapping probes. This step allows the identification of point mutations This step is followed by a second hybridizationarray thatallowsthe characterization of specific nutations by using smaller, specialized probe arrays complementary to all variants or natations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. In yet another embodiment, ay of a variety of sequencingreactions known in the art can be used to directly sequence one or more biomarkers gene of the invention, including a gene listed in Table I and the Examples, or a fragment thereof, and detect inutations by comparing the sequence of the sample biomarker gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natt Acad, Sci, USA 74:560 or Saingr (1977) Proc. Nat Acad Sci. USA 74:5463. It isalso contemplated thatany ofa variety of automated sequencing procedures can be utilized when performing the diagnostic assays(Naeve, C, W- (1995) Bintechniques 19:448-53), Including sequencing by mass spectrometry (sec, etg., PCT Intemational Publication No WO 94/16101; Cohen e al. (1996) Adv. Chromatogr. 36:127462;and Gtriffin etal (1993) Appl Biochem- Biotechno 38:147-159). Other methods for detecting mutaions inoneormorebiomarkersgeneofthe invention, including a gene listed in Table 1 and the Examples, or fragments thereof. include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myrs et al (1985) Science 230:1242) in general, the art technique of "mismatch cleavage"'starts by providingheteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained froma tissue sample, The double-stranded duplexes are treated with anagent which cleaves single-stmnded regions of the duplexsuch aswhich will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNADNA hy brids treatedwithSinucleaseto enzvmatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexs cantbe treatedvith hydroxylamine or osmiun tetroxideand vith piperidine inorder to digest mismatched regions. After digestion of the inismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation, See, for example, Cotton et al (1988) Proc. Nati. Acad. Sci. USA 85:4397 and Saleeba e al (1992)Methods Enznmol. 217:286-295. Inia preferred embodiment, the control DNA or RNA can be labeled for detection. In stillanotherembodiment, the mismatch cleavage reaction employs one ormore proteins that recognize mismatched base pairs in double-stranded DNA (so called"DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations inbiomarkergenes of the invention, including genes listed in Table I and the Examples, or fragments thereof obtained from samples of cells. For example, the mutY enzyme ofrE. coli cleaves A at G/A mismatchesand the thymidine DNA glvcosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al (1994) Carcinogenesis 15:1657-1662). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, ifany, can be detected from lectrophoresis protocols or the like, See, for example, U.S. Patent 5,459,039, In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in biomarker enes of the invention, including genes listed in Table I and the Examples, r fragnents tlireof. For example, single strand confornation polymorphism (SSCP) ma be used to detect differences inelectrophoreticniobility between mutant and. wild type nuclei acids (Orita e al (1989) Proc Nt. Acid. Seil USA 86:2766; see also Cotton (1993) Mutat. Res, 285:125-144 and Hayashi (1992) Gieet. Anal. Tech. Apple. 9:73 79). Single-stranded DNA fragments of sample and control nucleicacids will be denatured and allowed to reniture. The secondary structure of single-stranded nucleic acids varies accordingto sequence, the resulting alteration inelectrophoreic mobility enables the detection cif even single base change The DNA fragments may be labeled or detected with labeled probes, The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, thesubject method utilizes heteroduplex analysis to separate double stranded heeroduplex molecides on the basis ofchanges in electrophoretic mobility (Keen e ca. (1991) Trends Genet. 7:5). In vetanother embodiment the movement ofmutant orwild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers eiat (1985) Nature 313:495). When DOGE is used as the method of analysis, DNA vill bemodified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place ofa denaturing gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem 265:12753.). Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonuclentide hybridization, selective amplification, or selective primer extension. For example, nligonucleotide priners may be prepared in which the knownmIutation is placed centrally and then hybridized to target DNA under conditions which permit hybridizationonly ifa perfectmatch is found (Saiki cr al (1986) Nature 324:163; Saiki et al (1989) Proc. Natd. Acad. Sci, USA 86:6230). Such 'allele specific oigonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the yvbridizing membrane and hybridized with labeled target DNA. In some embodiments, the hybridization reactions can occur using biochips, microarrays, etc., or other array technology that are well known in the art. Ahternatively.allele specific amplification technology which depends on selective PCRamplification may be used in conjunction with the instant invention. Oligonuelectides used as primers for specific amplification may carry themutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs ei al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme Yend of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (I993) Tibtech 11:238) In additionit may be desirable to introduce a novel restriction site in the regionof the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol Cell Probes 6:1)- It is anticipated thatin certain embodiments amplification may also be performed using Taq ligase for aniplification (Barany (1991) Proc. Natt Acad, Sci USA 88:189). In such cases, ligaion I occur only if there is a perfect matchat the end of the 5' sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, byatilizingpre packaged diagnostic kits comprising at least one probe nucleic acid or anuibody reagnt described herein, which may be conveniently usede.g., in clinical settings to diagnose patients xhibiting symptoms orfFamily hisiorv of a disease or illness involving one or more bioniarkers of the invention, including one or more bioniarkers listed in Table I and the Examples, or fragments thereof 3,.Monitoring of Effects Durina Clinical Trials Monitoring the influence of agents(e.g drugs) on the expression oractivity of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples, or a fragment thereof (e.g, the modulation of a cancer state) can be applied.not only in basic drg screening, but also in clinical trials. For example, the efecivenssof an agent determined by a screening assay as described herein to increase expression and/or activity of one or more biomarkers of the invention, Including one or more biomarkers listed in Table 1 and the Examples or a fragment thereof, can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of one or more biomarkers of the invention including one or more biomarkers of the invention, includingone or more biomarkers listed inTable I and the Examples, or a fragment thereof, relative to a control reference. Alternatively, the effectiveness of anagent detenined by a screening assay to decrease expression and/or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examplcs, or a fragment thereof, can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of the binarker ofthe invention, including one or more biomarkers listed inTable I and the Examples or a fragment thereof relative to a control reference. In such clinicaltrials, the expression and/oractivity of the biomarker can be used as a"read out" or marker of the phnotype of a particular cell. In some embodiments, the present invention provides a method for monitoring the effectiveness of treatIment of a subject With an agent(e.gan agonist,antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of(i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii)detecting the level of expression and/or activity of one or more biomarkers of the invention,including one or more biomarkers listed in Table I and the Examples or fragments thereof in the preadministration sample (iii) obtain one or morepost-admistration samples from the subject; (iv) detecting thelevel ofexpression or activity of the biomarkerin the post administration samples; (v) comparing the level of expression or activity of the biomarker or fragments thereof in the pre-administration sample with the that of the biomarker in the post administration sampleorsamples; and. (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression oractivity of one or more biomarkers to higher levels than detected (ag, to increase theeffectiveness of the agent) Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of thle biomarker to lower levels than detected (eg to decrease the effectiveness of the agent) According,to such an embodiment, biomarker expression or activity may be used as an indicator of the effectiveness of an agent, even in theabsence of an observable phenotypic response.

D. Methods of Treatment The presentinvention provides for both prophylactic and therapeutic methods of treating a subject at risk of(or susceptible to)a disorder characterized by insufficient or excessive production of biomarkers of the invention, including biomarkers listed in Table 1 and the Examples or fragments thereof, which have aberrant expression or activity compared to a control. Moreover, agents of the invention described herein can be used to detect and isolate the biomarkers or fragments thereof, regulate the bioavailability of the biomarkers or fragments thereof, and modulate biomarker expression levels oractivity. 1. Prophylactic Methods In one aspect, the invention provides a method for preventgin a subject, a disease or condition associated with in aberrant expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the.Examples or a fragment thereof, by administering to the subject an agent which modulates biomarker expression or at least one activity of the biomarker, Subjects at risk fora disease or disorder which is caused or contributed to byaberrant biomarker expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein, Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthebiomarker expression or activity aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. 2. Therapeutic Methods Anotheraspect ofthe invention pertains to methods of modulating the expression or activity or interaction with natural binding partners) of one or more biomarkers of the invention, including one or more biomarkers listed in Table I and the Examples or fragnients thereof, for therapeutic purposes, The biomarkers of the invention have been demonstrated to coruteithcancer (e.g head, aeck and/or lung cancers), Accordingly, the activity and/or expression of the biomarker, as well as the interaction between one or more biomarkers or afragment thereof and its natural binding partners) or a fragment(s) thereof can be modulated in order to modulate the inmne response, Modulatory methods of the invention involve contacting a cell with one or more biomarkers of the invention, including one ormore biomarkers of the invention, icluding one or more biomarkers listed in Table 1 and the Examples or a fragment thereof or agent that modulates one or more of the activities of biomarker activity associated with the cell In some embodiments, the bionarkers are or encode secreted molecules such that contacting a cell with one or more biomarkers of theinvention or agent that modulates one or more of the activities of biomarker activity isunnecessary and contact vith a bodily fluid (e.g., blood, seruninng pleural fluid, etc.) is sufficient. An agent thatmodulates biomarker activity can be an agent as described herein, such as a nucleic acid ora polypepride, a naturally-occurring binding partner of the biomarker, an antibody against the biomarker. a combination of antibodies against the biomarker and antibodies against other imm1ne related targets, one or more biomarkers agonist or antagonist, a peptidomimetic of one or more biomarkers agonist orantagonist, one ormore biomarkers peptidomimetic, other small molecule, or small RNA directed against oramimic of one ormore biomarkers nucleic acid gene expression product An agent that modulates the expression of one or more biomarkers of the invention, including one or more biomarkers of theinvention,including one or more biomarkers listed in Table I and the Examples or a fragment thereof is, e.g., antisense nucleic acid molecule, RNAi molec, shRNA, maturemniRNA, pre-miRNA, pri-miRNA, riRNA*, anti-miRNA, or a miRNA binding site, or variant thereof, or other small RNA molecule, triplex oligonucleotide, ribozyme or recombinant vector for expression of one or more biomarkers polypeptide. For example, an oligonucleotide cnmplmcntary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized, One or more antisense oligonucleotides canbeadded to cell media, typicallyat 200pg/ml, or administered to a patient to prevent the synthesis ofone or more biomarkers polypeptide, Theantisense oligonucleotide is taken up by cells and hybridizes to oneor more bioarkers mRNAtoprevent translation.Aternativlyanogonuleotidewicbindsdouble stranded DNA to form a triplex construct to prevent DNA unwindingand transcription can be used. Asa result of either, synthesis of biomarker polypeptide isblocked.When biomarker expression is modulated, prefeiably, such modulation occurs by a means other than by knocking out the biomarkergene. Agents which modulate expression, by virtue of the fact that they control the amount of bionarker in a cell, also modulate the total amount of biomarkeractivity ina cell In one embodiment, the agent stimulates one or more activities of one ormore biomarkers of the invention, including one or more biomarkers listed in Table 1 and the Examnpesorafiagmentthereof, samples of such stimulatoiy agents include active biomarker polypeptide or a fragment thereof and a nuleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into the cell (eg,,eDNA, mRNA, shRNAs, siRNAs. small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA* anti mniRNA, or a miRNA binding site, or a variant thereof, or other fmnctionally equivalent molecule known to a skilled artisan). In another embodiment, the agent inhibits one or more biomarker activities. in one embodiment, the agent inhibits or enhances the interaction of the biomarker with its natural binding partnerss. Examples of such inhibitory agents include antisense nucleic acid molcculcs, anti-bionarkerantibodies, biomarker inhibitors, and compounds identified in the screening assays described herein. These modulatory methods can be performed in vitro (e.g., by contacting the cell with the agent) or, alternatively., by contacting an agent with cells in 1vo (eg, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with condition or disorder that would benefit from up- or down-modulation of one or more biomarkers of the invention listed in Table I and the Examples or a fragment thereof, eg., a disorder characterized by unwanted, insufficient, or aberrant expressionoractivity of the biomarker or friagments thereof In one embodiment, themethodinvolvesadministeringanagent (eg.,an agent identified by a screening assay described herein), o comination ofagents that modulates (e.g upregulates or downregulates) biomarker expression or activity. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or acvity Stinulation of biomarker acivity is desirable insituations in which the bionarkeris abnormally downregulatedand/orin which increased biomarker activity is likely to have a beneficial effect. Likewise, inhibition of biomarker activity is desirable in stations in which hiomarker is abnormally upregulated and/or in hich decreased biomarker activity is likely to have beneficial effect in addition, these modulatory agents can also be administered in combination therapy with, egchemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or withsurgery, cryotherapy, and/or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g., standard-of-care treatments for cancer well knovnto the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, these modulatory agents can be administered with a therapeutically effective dse of chemotherapeutic agent. In another embodiment, these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers- The dosing reimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer (e.g, head, neck, and/or lung cancers), being treated, the extent ofthe disease and other factors familiar to the physician of skill in theart and can be determined by the physician, In some embodiments, it is useful todow regulate immune responsesin other embodiments, it is useful to upregulate immune responses a. Downregulation ofimmuneResonsesby Modulation There are numerous embodiments of the invention for upregulating theinhibitory functionor downregulatng the costimulatory functionof a PD-1 ligand (e.,solublePD Li) townhreowl inrgatmne responses. Dowregulation can bein the form of inhibiting or blockingan immune response already in progress, or may involve preventing the induction of an immune response. The functions of activated immune cells can be inhibited by down-regulating immune cell responses, or by inducing specific anergy in immune cells, or both. For example, the immune response can be downmodulated.using PD- ligand. (ag, soluble PD-L1) polypeptides(eg, soluble, monomeric forms of a PD ligand (e.g soluble PD-L1) polypeptide such as PD-1 ligand (e.g, soluble PD-LI)-g) and/or anti-PD-I ligand (eg.soluble PD-L1) antibodiecs that block the interaction of PD-I ligand (e.g., soluble PD-LI) with B7 polyeptidt (egwhile not affecting or increasing the interaction between PD-Li and PD-1) or which promote the binding of a PD- Iigand (e.g, soluble PD-LI.) with PD-1, (e.gwhile not affecting or while inhibiting the Interaction between a B7 polypeptide and the PD-1 ligmnd (e.g., soluble PD-LI)). Other exemplary agents which can block these interactions include anti-B7 polypeptide, a B7 polypeptide, or a blocking small molecule. Inaddition, in embodiments where PD-L Ibinds toan inhibitory receptor, forms of PD-IJ. that bind to the inhibitory receptor, multivalent PD-I. on a cell surface, can be used to downmodulate the immune response. Likewise,the PD-i pathway can also be stimulated by the use of an agent thereby downmodulate the inune response. Inhibition ofthe interaction ofB7-4with a stimulatoryreceptor on an immune cell (eg., by using a soluble fbri of PD-1 and/or CTLA4) oractivation of PD-1 (e.g, usingan activating antibody which cross-links PD-i) may provide negative signals to immune cells, Agents that promote binding of a PD-Iligand (e.g, soluble PD-L) to PD-I or a B7 polypeptide to CTLA4. while not affecting or reducing the binding of the PD- ligand (e.g, soluble PD-1) to the B7 polypeptide, can also be used to down modulate the immune response. Exemplary agents include PD-I peptide mimetics, identified by the methods described herein. In one embodiment of the invention, an activating antibody used to stimulate.PD-1 activity is a bispecific antibody. For example, such an antibody can comprise a PD-1 binding site and another binding site which targets a cell surface receptor on an imun ecell, e.g, on a Tcell, a B cell, oramycloid cell. Inoneembodiment, such an antibody, inaddition to comprising a PD-Ibinding site can further comprise a binding site which binds to a molecule which is in proximity to an activating or inhibitoryreceptor, e.g, B-cell antigen receptor, a T-cell antigen receptor, or an Fe receptor in order to target the molecule to a specific cell population. For example, a CD3 iantigen, a T-cell receptor chain, LFA-1, CD2, CTLA-4, immunoglobulin, B cell receptor, Ig alpha, Ig beta, CD22, or Fe receptor could be used. Such antibodies (or other bispecific agents) areart recognizedand can be produced, e.gasdescribedherein. Selection ofthis second antigenfor thehbispecific antibody provides flexibility in selection of cell population to be targeted for inhibition. In another embodiment, the co-ligation of PD-I and an activating orinhibitory receptor on a cell can enhance the generation of a negative signal via PD-1. Such co ligation can be accomplished .g, by use of a bispecific agent, e.g, a bispecific antibody as described herein having specificity for both PD-1 and a molecule associated with a receptor. In another embodiment, the use of a multivalent form ofan agent that transmits a negative signal via PD-1 can be used to enhancethe transmission of anegative signal via PD-1, egan agentpresented on a bead or on a surface. In anotherembodiment, a such a multivalent agent can comprise two specificities to achieve co-ligation of PD-i and a receptor or a receptorassociated molecule (eg. a bead comprising anti CD3 and PD-L). Agents that block or inhibit interaction of PD-L Iwith a costimulatory receptor (e.g, soluble forms of PD-L or blocking antibodies to PD-L) as well as agents that promote a PD-LI .- mediated inhibitory signal or agonists of PD-i which activate PD-I (eg. PD-I activating antibodies or PD-l activating small molecules) can be identified by theirability to inhibit immune cell proliferation and/or effector function or to induce anerg when added to an in vitro assay. For example, cells can cultured in the presence of an agent that stimulates signal transduction via an actuvating receptor. A number ofart recognized readouts of cell activation can be employed to measure, eg, cell proliferation or effector function (e.g, antibody production, cytokine production, phagocytosis) in the presence of the activating agent. The ability of a test agent to block this activation can be readily determined by measuring the ability of the agent to effect a decrease in proliferation or effector function being measured. In one embodiment of the invention, tolerance isinducedagainst specific antigens by co-administeringan antigen withan agent (eg antibody, peptide fusion protein, or small molecule) which blocks the interaction between a PD-1 ligand (eg, soluble PD-Li) and a B7 polypeptide. For example, tolerance can be induced to specific proteins. In one embodiment, immune responses to allergens, or toforeign proteins to which an immune response is undesirable, can be inhibited. For example, patients that reeve Factor VIII frequently generate antibodies against this clotting factor. Co-adinistration ofan agent that blocksa.PD-l igand( soluble PD-I.)-mediated costimulator signor an agent that stimulates a PD-I mediatedinhibitory signalin combination with recombinant factor VIII (or by physically linked to Factor VIlI, e.g, by cross-linking) can result in downmodulation. In one embodiment, fusion proteins comprising a firstPD-ligand (e.g,soluble PD-LI) peptide fused to a second peptide can be used t block the interacion of the PD-1 ligand (e.g, soluble PD-Li) with a B? polypeptide on an immune cell, to thereby downmodulatc immune responses. In one embodinent, the second peptide blocks an activity of'another B lymphocyteantigen (e3g, B7-1, B7-2, or B7-3) to further downmodulate immune responses. Alternatively, two separate aents that downmoduate immune responses can be combined as a single composition or administered separately (simultaneously or sequentially) to more effectively downregulate immune cell mediated immune responses in a subject, For instance, a PD-1 ligand (e.gsoluble PD-LI) can be combined with a B7 polypeptide, or with a combination of blocking antibodies (e.g, antibodies againstPD-1 igand (e.g, soluble PD~1N) polypeptide with antiB7-1 and/or anti-B7-2 monoclonal antibodies)- Furthermore, a therapeutically active amOunt of one or more of the subject agents, canbeusedinconjunction with other dovnmodulating reagents to influence immune responses. Examples oftother immunoniodulating reagents include, without linittina, antibodies that block a costimulatory signal, (e.g., against CD28 or ICOS), antibodies thatactas agonists of CTIA4, and/or antibodies against otherimmune cell markers (.v, against CD40, against CD40 ligand, or against cytokines), fusion proteins (e.g, CTLA4-Fc), and imnosuppressive drugs, (e.g rapamycin, cyclosporine A or FK506).

The agents described herein can also be useful in the construction of therapeutic agents which block immune cell function by destruction of cells. For example, portions of a PD-L Ior PD-I polypeptide can be linked to a toxin to make a cytotoxic agent capable of triggering the destruction of cells to which it binds. For making cytotoxic agents, polypeptides of the invention may be linked, or operatively attached, to toxins using techniques that are known in the art, e.g., crosslinking or via recombinant DNA techniques. The preparation of immunotoxins is,in general, well knownin theart(see.gU.S Pat Nos. 4,340,535, and EP 44167, both incorporated herein by reference). Numerous types of disulfide-bond containing linkers are known which can successfully be employed to conjugate the toxin moietykwith a polypeptide. In one embodiment, linkers that contain a disulfide bond that is sterically "hindered" are to be preferred, due to their greater stability in vivo, thus preventing release of the toxin moiety prior to binding at the site of action. Wide variety of toxins are known thatmay be conjugated to polypeptides or antibodies of the invention. Examples include:numnerous useful plant-., fungus- or even bacteria-derived toxins, which, by way of example, include various A chain toxis, particularly ricin A chain, ribosomeinactivating proteins such as saporin or gelonin, .aipha.-sarcin, aspergiilin, restrictocin, ribonucleases such as placental ribonuclese, angiogenic, diphtheria toxin, and pseudomonas exotoxin, etc. A preferred toxin moiety for use in connection with the invention istoxin A chain which has been treated to modify or remove carbohydrate residues, deglycosylated A chain. (U.S. patent 5,776,427) Infusion of one or a combinationofsuch cytotoxicagents (eg.,PD-Li ricin (alone or in combination with B7-2-ricin or B7-i-ricin), into a patient may result in the death of immune cells, particularly inlightof the fet thatactivatedimumne cells that express higher amounts of PD-Ll ligands. For example, because PD-l is induced on the surface of activated lymphocytes, anantibody against PD-i can be used to target the depletion of these specific cells by F-R dependentmechanisms or by ablation by conjugating a cytotoxic drug (ecg, ricin, saporin, or calicheamicin) to the antibody. In one eimbodiment, the antibody toxin can be a bispecific antibody. Such bispecific antibodies are useful for targeting a specific cell Population, e.g, usinga marker found only ona certain type of cell. e.g, a TCR, BCR, or FcRmolecule. Downregulating or preventing a PD-1ligand (e-g., soluble PD-Ll) interaction with a 137 polypeptide, or promoting an interaction between a PD-] ligand (e.g soluble PD-L1)

-11 2- and PD-1 (for example, withoutmodulating, or byadditionallyenhancing) the interaction between the PD-1 ligand (e.g, soluble PD--1) and the B7 polYpeptide (eg by stimulation of the negative signaling function of PD-1) is useful to downmodulate the immune response, e, in situations of excess inflammation; in tissue, skin and onan transplantation; ingraft-versus-host disease (GVHD); or in autoimmune diseases such as systemic upus erythematosus, and multiple sclerosis. For example, blockage ofimmune cell function results in reduced tissue destruction in tissue transplantation- Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by immune cells, followed by an immune reaction that destroys the transplant. The administration of a polypeptide wviich inhibits or blocks interaction of a PD-1 ligand (eg, soluble PD-1I) with a B7 polypeptide (such as a soluble, monomeric form of the PD-1 ligand (eg, soluble PD~Ll) or PD-1), alone orin conjunction with another downmod.ulatory agent, prior to or at the time oftransplantation can promote the generation of an inhibitory signal.Moreover, inhibition of PD-I1ligand (eg., soluble PD-Li) costimulator signals, orpronotion ofa PD-1 ligand (ag, soluble PD-L 1) or PD-1 inhibitory signals, may also be sufficient to anergize the immune cells, thereby inducing tolerance in a subject, Induction of long-term tolerance by blocking a PD-i ligand (e-g., soluble PD-L1) mediated costiulatory signalma avidthenecessityofrepeated administration of these blocking reagents. To achieve sufficient imnunosuppression or tolerance in a subject, it mayalsobe desirable to block the costimulatory function of other polypeptides. For example, it may be desirable to block the function of 87-1 and PD-Li; B7-2 and PD-L; B7-1 and 97-2 and PD-Ll; B7-1; B7-2; or B7-1 and 7-2 by administering a soluble form of a combination of eptids having an activityofeachoftheseantigens, blocking antibodies against these antigens or blocking small molecules (separately or together in a singlcComposition) prior to orat the time of transplantation.Alernaively, it may be desirable to promote inhibitory activity of a PD1 ligand (eg, soluble PD-LI) or PD-1 and inhibit a costimulatory activity of 17-1 and/or B7-2- Other downmodulatory agents that can be used in connection with the downmodulatory methods of the invention include, for example, agents that transmit an inhibitorysignal via CTIA4,soluble forms of CTLA4, antibodies that activate an inhibitory signal via CTLA4. blocking antibodiesagainst other immune cellmarkers or soluble forms of other receptor ligand pairs (e.g. agents that disrupttheinteractionbetween CD40 and CD40 ligand (cg, anti CD40 ligand antibodies)), antibodies against cytokines,

-11 3- orimmnosuppressivedrugsInanother embodiment, a combination ofat least two different PD-L1Iantibodies can be administered to achieve optimal bockingactivt. Downmiodulation of immune responses are also useful in treating autoiriune disease. Many autoimm1e disorders are the result of inappropriate activation of immune cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathologyof the diseases. Preventing the aivation of autoreactive immune cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of immune cells by disrupting interactions between PD Iligand (egsoluble PD-LI) and B7 polypeptides, or by promoting the interaction between PD-i ligand (eg,soluble PD-LI) and PD-1, without modulating or while downmodulating the interaction between PD-1 ligand (e.g. soluble PD- I1) and a B7 polypeptide are useful for inhibiting immune cell activation and preventing production of autoantibodies or cytokines which may be involved in the disease process, Additionally, agents that promote an inhibitory function of a PD-1 ligind (g-, soluble PD-L I) or PD-1 may induce antigen-specific tolerance ofautoreactive immune cells, which could lead to long-tern relief from the disease, The efficacy of reaents in preveting or alleviating autmmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases. Examples include marine experimental autoimnmune encephalis, systemic lupus erythematosus in MRLlpr//pr mice or NZB hybrid mice, murine autoimmune collagenarthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia graves (see, e.g, Pau edF immunology, Raven Press, New York, Third Edition 1993, chapter 30) Inhibition of imnmne cell activation isuseful therapeutically in the treatment of allergy and allergic reactions, e.g, by inhibiting IE producion.Anagentthat promotes a PD-1 ligand (eg., soluble PD-Li) or PD- inhibitory function can be administered toan allergicsubject to inhibit immune cell mediated allergic responses in the subject. inhibition of PD-i ligand (eg, soluble PD-LI) costimulation of imname cells or stimulation of a PD-i ligand (e.g., soluble PD-LI) orPD-1 inhibitory pathway can be accompanied by exposure to allergen in conjunction withappropriate MHC polypeptides. Allergic reactions can be systemic or local innature, depending on theroute of entry of the allergenand the pattern of deposition of IgE onmast cells orbasophils. Thus, inhibition of immune cell mediated allergic responses locally or systemically by administration ofan inhibitory form of an agent that inhibits the interaction of a PD-I ligand (e.gsoluble PD-LI) with a costimulatory receptor, oran agent that promotes an inhibitory finetion of a PD-I ligand (eg,,soluble PD-L1) or PD-I. Inhibition of immne cell activation through blockage of the interaction of a PD-I ligand (e.g, soluble PD-LI) and a B7 polypeptide or through promotion of the interaction between a PD-I ligand (e.g, soluble RD-L) and PD-1, without modulating or while downmodulating the interaction between the PD-l ligand (e.g, soluble PD-Ll)and a B7 polypeptide may also be important therapeutically in iriinfections of immune cells. For example, in the acquired immune deficienysyndrome (AIDS), viral replication is stimulated by immune cell activation. Modulation of these interactions may result in inhibition of vir-l replication and thereby ameliorate the course of AIDS. Modulation of these interactions nay also be useful in promoting the maintenance ofpregnancy. PD-1 ligand (e.g.I solble PD-L I) is normally highly expressed in placental trophoblasts, the layerofcells thatformsthe interfacebetweenmotherand fetusandmayplayarole in preventing maternal rejection of the fetus. Females at risk for spontaneous abortion (eg, those who have previously had a spontaneous abortion orthose who have had d'iculty conceiving) because ofimmunuologic rejection of the enibryo or fetus can be treated with agents that modulate these interactions. Downregulation of an immune response by modulation of PD-i ligand (e.g soluble PD-Li)/ B7 polypeptide, binding or by modulation of PD-i ligand (e,g, soluble PD-L.1)/ PD-1 bindmoinav also beuseful in treatingan autoimmune attack of autologous tissues. For example, PD-I ligand (e.g., soluble PD-L) is normally highly expressed in the heart and may protect the heart from autoimmune attack. This is evidenced by the fact that the Balb/c PD-i knockout mouse exhibits massive autoimmune attack on the heart with thrombosis. Thus, conditions that are caused or exacerbated byautoimmune attack (e.g. in this example, heart disease, myocardial infraction or atherosclerosis) may be ameliorated or improved by modulation of these interactions. It is therefore within the scope of the invention to modulate conditions exacerbated by autoimmuneattack, such as autoimmune disorders (as well as conditions such as heart disease,myocardial infarction, and atherosclerosis). b. Upreuulation ofIrune Responses Also useful therapeutically is the blockage of the interaction of a PD- ligand(e.g, soluble PD-LI) with PD-1, and/or a B7 polypeptide with CTLA4, without modulating or while upregulating the interaction between the B7 polypeptide and the PD- Iligand (e,g_ soluble PD-LI), or by promoting the interaction of the PD-Iigand(e.,soluble PD-LI) withthe B7 polypeptide (eg_ while not affecting or while inhibiting the interaction between the PD-1 ligand (e.g soluble PD-L1) and PD-1) asia means ofupregulating an immune response. Blocking the interaction between a B7 polypeptide and a PD-1 ligand (e.g, soluble PD-LI) to thereby increase the interaction between the B7 polypeptide and CD28, is also useful toupregulateimmune responses Upregulation of immune responses can be in thefonn of enhancing an existing immune response or eliciting aninitialimmune response. For instance, enhancing an immune response using the subject compositions and methods is useful in cases of infections with microbes (e.g, bacteria, viruses, or parasites) In one enbodimentan agent thit blocks the interaction ofa PDIl ligand (e.g soluble PD Li) with PD-, without modulating or while upregulatingthe intirconbetweenaB7 polypeptideandthePD-iligand ( olublePD-L)orbyprom ttheinteractionof the PD-i ligand (e.g, soluble PD-LI) with theB7 polypeptide, is used to enhance the immuneresponse.Such an agent(e.ganonactivatingantibodythatblocksPD-LI binding to PD-1) is therapeutically useful in situations where upregulation of antibody and cell-mediatedresponses would be beneficial. I a preferred enibodiment, the agent inhibits the interaction between. PD-1 anda PD-I ligand (e.g.soluble PD-LIl), without inhibiting the interaction betweenthe PD-1 ligand(e.g, soluble PD-LI) and a B7 polypeptide (e.g, an interaction which prevents PD-L Ifrom binding to PD-1). Exemplary disorders include viral skin diseases, such as Herpes or shinglesin which casesuch an agent can be delivered topically to theskin. In addition, systemic viral diseases such asinfluenza, the common cold, and encephalitis might be alleviated by systemic administration of such agents. Alternatively, immune responses can be enhanced in aninfected patient through an er vivo approach, forinstance, by removing immune cells from the patient,contacting immune cells in viro withanagent that blocks the interaction of a PD-1 ligand (e.g, soluble PD-LI) with PD-1,withoutniodulating or while pmodulating the interaction between a B7 polypeptide and the PD-1 ligand (e.g, solublePD-L I), or by promoting the interaction of the PD-1 ligand (e.g., soluble PD-Ll) with the B7 pnlypeptide., and reintroducing the in vitro stimulated immune cells into the patient. In certaininstances,itmay be desirable to further administer other agents that upregulate immune responses, for example, forms of other B7 family members that transduce signals via costimulatoryreceptors, in order to further augment the immune response. An agent that blocks the inractionof a PD-1 ligand(e.g.solublePD-L)withPD I (e.g, withoutmodulating or while upmodulating the interaction between a B7 polypeptide and the PD- Iligand (eg, soluble PD-L1) or by enhancing the interaction of ihe PD- igand (eg. soluble PD-L1) withdieB7 p plypp-ide) can be used prophylactically in vaccnes againstvarious polypeptides (ag polypeptides derived from pathogens). Immunity against a pathogen (cg, a virus) can be nduced by vacinating with a viral protein along with an agent that blocks the interaction of a PD1 ligand (e.g soluble PD-L1) with PD-1, without odulating or while upmodulating the interaction between a B7 polypeptide and the PD-1 ligand soluble or by promoting the interaction of the PD-I igand (c.g, soluble PD-L) with the B7 polYpeptide, in anappropriateadjuvant. In another embodiment, upegulation or enhancement of animmune response function, as described herein, is useful in the induction of tunor immunity In another embodiment, the immune response can be stimulated by the methods described herein, such that preexisting tolerance is overcome. For example., immune responses against antigens to which a subject cannot mount a significant immune response e.g, utaatologus antigen, such as a tumor specific antigens can be induced by administerim an agent that blocks the interaction of a PD- Iigand (eg., soluble PD-Ll) withPD- (eg, vhout modulating or while upnodulating the interacion between a B7 polypeptide and te PD- ligand (e.g. soluble PD-L 1) or by promoting the interaction of the PD- ligand (e.g. soluble PD-LI) itb the B7 polypeptide). In one embodiment, a soluble PD-I or a soluble PD-1 ligand (eg, soluble PD-L1) that inhibits the interaction ofa PD- ligand (g, soluble PD-LI) with PD-1, without niodulating or while upmodulating the interaction betweenaB7 polypeptideand the PD-1 Ligand (eg, soluble PD-LI), or by promoting the interaction of the PD- ligand (eg, soluble PD-L1) with the B7 polypeptide, can be used to enhance an immune response (eg, to a tumor cell). In oneembodiment, an autologous antigen, such as a tumor-specific antigen can be coadministered. In another emubodimenzhesubject agents can be used as adjuvants to boost respoinses to foreign antigens in the process of active immunization. In yet another embodiment, the production of a form of PD-L that binds to an inhibitory receptor or that competes with the binding of PD-Li to a costimulatory receptor (eg,a form of PD-LI that binds to PD-1 or a naturally occurring soluble molecule) can be inhibited, eg., using antisense RNA, in order to upregulate theimmune response. For example, none embodiment, the production of inhihitorv PD-L molecules by a tumor cell can be inhibited in order to increase anti-tumor immunity. In one embodiment, immune cells are obtained froma subject and cultured ex vivo 5 in the presence of an agent as described herein., to expand the population of immune cells and/or to enhance immune cell acivation. In afurther embodiment theimmune Cells are then administered to a subject. Immune cells can be stimulated in viro by, for example, providing to the immune cells a primary activation signal and a costimuiatory signiiaL as Is known in the art. Various ages can also be used to costimulate proliferation of immune cells. In one embodiment immune cells are cultured e vivo according to the method described inPCT Application No. WO 94/29436. The costimulatory polypeptide can be soluble, attached to a cell membrane, or attached to a solid surface, such as a bead.

IV. Pharmaceutical Compositions Inanotheraspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeuically-effective amount of an agent tha modulates (e. Increases or decreases) PD-1, membrane-bound PD-LI, and/Or soluble PD-LI levels, formulated together With one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the 2 present invention ma be specially formulated for administration in solid or liquid form, including ihose adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, bonuses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solun for suspension; (3) topical application, for examples a cream, ontmient or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary .cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the conipound. The phrase "therapeutically-effectiveountas used herein means that amount of anagent that modulates (eg., inhibits) PD-1, membrane-bound PD-LI, and/orsoluble PD Li levels, or expression and/or activity of the receptor/igand complex, orcomposiion comprisganagentthatnoduaes(., iibts)PD-.meibrane-bound PD-Li, and/or soluble PD-Ll levels, or expression and/oractivity of the receptor/igand complex, which is effective for producing some desired therapeutic effect, e.g cancer treatment, ata reasonable benefit/risk ratio. The phrase "pharnaceutically acceptable"is employed herein to refer to those agents, materials, compositions, and/or dosageforms which are, within the scope ofsound medical judgment, suitable for usein contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase pharmaceuticlacceptecanier" as used herein means a pharniacutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemicalfrom one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients ofthe formulation and not injurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) celhdlose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, stich as cocoa butterand suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oiL corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene Olvcol; (12) esters, such as ethyl oleate and ethyl laarate; (13) agar; (14) buffering agents, such as magnesium hydroxide andalutminum hydroxide; (15) alginic acid; (16) pyrogen-freewater; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions and (21) other non-toxic compatible substances employed in pharmaceutical formulations. The term pharmaceutically-acceptablesats"refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates( inhibits) RPD-1, iemibrane-boind PD-Ll, and/or suhible PD-L. levels, or expessioandoiattvityof the receptor/ligand complex encompassed by the invention. These salts can be prepared in situ during the final isolation and purification of the respiration ncouplingagentsor by separately reacting a purified respiration uncouplng agent inits free base form with a suitable organic or inorganic acid, and isLating the salt ths formed.. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, naleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate and laurvlsulphonate salts and the like (See, for example, Berge etaL (1977) 'Pharmaceutical Salts",1J Pharn Sci. 66:1-19) In other cases, the agents useful in themethods of the present invention may contain one or more acidic functional groups and, thus are capable of forming pharmaceutically acceptablesalts with pharmaceutically-acptbleases. The term"pharmaceutically acceptable salts"in these instances refers to the relativelynon-toxic, inorganic and organic base addition salts of agents that modulates (eg, inhibits) PD-1, membrane-bound PD-LI, and/orsoluble PD-L I levels, orexpression and/or activityof the receptor/ligand complex. These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respirationuncoupling agent in its free acid formnwih a suitable base, such as the hydroxide, carbonatenr bicarbonate of apharmaceutically-acceptable metal cation, with ammonia, or with a phannaceuticall-acceptable organic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,ragnesturn., and aluminum salts and the like. Representative organicarmines useful for the formation of base addition salts includeethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et a,spra) Wetting agents, emulsifiers md lubricants, such as sodium laryl sulfateand magnesin stearate, as well as coloring agents, release agents,coating agents, sweetening! flavoring and perfumingagents, preservatives and antioxidants can also be present in the compositions. Examples ofpharmaceutically-acceptablantioxidants include:([) water soluble antioxidants, such as ascorbic acid, cystene hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants.,such as ascorbyl palmitate, butylated hydroxyanisole (BRA), butylated hydroxytolene (BHT), lecithin, propyl gallate, alpha-tocopheroland the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol tartaric acid, phosphoric acid, and the like. Formulations useful in the methods of the presentinvention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vagimal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage

- i20 - formi and may be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carter material to produce a single dosageformwillvarydependingupont host being treated, the particular modeof administration. The amount ofactive Mredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 % toabout 99% of activeingredient, preferably fromabout5% to about 70%, most preferably fromabout10% to about 30%. Methods of preparing these fornulanions or compositions include thestep of bringing into association an agent that modulates (e.g. increases or decreases) PD-1, membran-ebound PD~1, and/or soluble PD-L1 levels, with the carrier and, optionally, one or more accessory inrredients, In general, the formulations are prepared by uniformly and intimately bringing mtci association a respiration uncoupling agent with liquid carriers, or finely divided solid carriers, or both,and then, if necessary, shaping theproduct Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacath), povders, granules, or as a solution or a suspension in an aqueous or non aqueous liquid, or as an oil-in-water or water-inoilliquid emulsion,or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste. insoliddosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceuticaly-aceptablecarriers, such as sodium citrate or dicalciam phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrroidone, sucrose and/or acacia; (3)humectants, such as glycerol (4) disinteratinantssuch as agar-agar. calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate: (5) solution retardingagents, such as paraffin; (6) absorption accelerators, such asquimernary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a tale, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof;and (10) coloring agents, In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hardfilled gelatincapsules using such excipients as lactose ormilk sugars, as wellas high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets maybe made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert hquid diluent, Tablets, and other solid dosage formssuch as dragees, capsules, pills and granules, may optionlly be scored or prepared with coatings and shells, such as enteric coatings and other coatings we] Iknown in thepharmaceutical-formulatigart. They may also be formidated so as to provide slow or controlled release oftw active ingredient therein using for cxauple ydiroxypropylmethil cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres, They may be sterilized by, for example, filtration through a bacteriaretainingfilter, or by incorporating steriizing agents in the form of sterile solid compositions, which can be dissolved In sterile water, or some other sterile injectable medium immediately beforeuse. These compositions may also optionally contain opacifyingagents and may be of a composition that they released he active ingredient(s) only, or preferentially, ina certain portion of the gastrointestinaltract, optionally, in a delayed manner. Examples of embedding compositions, which can be used include polyineric subsctancs and waxes. The active ingredient canalso be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration includepharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions,symps and elixirs.In addition to the active ingredient the hquid dosage forms may contain inert diluents conmionly used in the art, such as, for example, wateror other solvents,soubilizingagentsandeulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glecol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof Besidesinert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof, Formulationsfor rectal or vginial administration may be presented asa suppository, which may be prepared by mixing onl or more respiration uncoupling agents with one or more suitable nonirritating excipiso carrierscomprising,frexample, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and release the active agent. Fornulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate, Dosageforms for the topical or transdermal administration of an agent that modulates (eg increases or decreases) PD-1, membrane-bound PD-Li, and/or soluble PD L Levels include powders, sprays, ointments, pastes, creams lotions, gels,solutions patchesand inhalants. The active component may be mixed under sterile conditions with a phanaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such animal and vegetables, oilswaxes,paraffins, starch, tragacanth, celldose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide,ormixtures thereof. Powders and spras can contain, in addition to an agent that modulates (eg, increases or decreases) PD-1, meibrane-bound PD-LI. and/or soluble PD-L Ilevels, excipients such as lactose, tale, silicic acid., aluminum hydroxide, calcium silicates and. polyamnide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chloroluiorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane, The agent that modulates (e.g, increases or decreases) PD-1,membrane-bound PD .1, and/or soluble PD-L levels, can bealternatively administered byaerosol This is accomplished by preparingan aqueous aerosol, liposomal preparation or solid particles containing the cornpound. A nonaqucous (e.g, fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound. Ordinarilly an aqueous aerosol is made by formulating an aqueous solution or 1) suspension oftheagent together with conventional pharmaceutically acceptable can-iers and stabilizers. The carriersand stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Twcens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitin esters, oleic acid., lecithin, amino acids such as glycine, buffers, salts, sugars or sugaralcohols. Aerosols generally are prepared from isotonic solutions. Transdernal patches have the added advantage of providing controlled delivery of a respiration uncoupling agentto the body, Such dosage tbrms can beimade by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimtic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane ordispersingthe peptidomimetic in a polymer matrix or gel. Ophthalmic formlUlations, eye moments, powders, solutions and the like,are also contemplated as being within the scope of this invention, Pharmaceutical compositions ofthis invention suitable for parenteral administration comprise one or more respiration uncoupling agents in combination with one ormore phariaceutically-acceptable sterile isotonic aqueous or nonaquous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formlation isotonicwiththe blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqucous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsiyingagents and dispersing agents- Prevention of the acting of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents,for example, paraben, chlorobutanol, phenol sorbic acid, and the like.it may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.Inaddition,prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delayabsorption such asaluminum monostearate and gelatin, In some cases, in order to prolong the effect ofa drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquidsuspension of crystallineor amorphous material having poor water solubility, The rate of absorption of the drug the p depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline tbrm, Alternatively, delayed absorption ofaparenterallyadministered drugform is accomplished by dissolving or suspending the drug in an oilvehicle, injectabedepotforms are made by forming microencapsule matrices of an agent that modulates (e.g., increases or decreases) PD-1, membrane-bound PD-Li, and/or soluble PD-L1 levels, in biodegradable polymUs such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, whichare compatible with body tissue. When the respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0. 1to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration,without being toxic to the subject. The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by; for example, intravenous inechtin, local administration (seeUS. Pat, No 5,328,470) r by stercotactic injection (see e g, Chenet al, (1994) Proc. NatI. Acad. Sci. USA 91:3054 3057), The pharmaceutical preparation of the gene therapy vector can Include the gene therapy vector in an acceptable diluent, or can comprise a slow release iatrix in which the g.ne delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e-g retroviral vectors, thepharmaceutical preparation can include one or more cells which produce thegene delivery system.

V. Administration of Agents The cancer diagnostic, prognostic, prevention, and/or treatment modulating agents of the invention are administered to subjects in a biologically compaible form suitable for pharmaceutical administration in vivo, to either enhance or suppress immune cell mediated immune responses. By "biologically compatible form suitable administration invivo" is meant a form of the protein to be administered in which any toxic effects are outweighed by hthetherapeutic effects of the protein. Theterm"subjet is intended to include living organisms in which an immune response can be elicited, eg, maninals, Examples of subjects include humans, dogs, ats,mice, rats, and transgenic species thereof. Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone orincombination with a pharmaceutically acceptable carrier. Administration of a therapeutically active amount of the therapeutic composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, a therapeutically active amount of a blockingantibody may vary according to factors such as the disease state, age, sex, and weight of thc individual, and theability of peptide to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation,

The agents of the invention described herein can be administered in a convenient mannersuch as byinjection (subcutaneous intravenous,etc.),oraladministration inhalation, transdermnal application, or rectal administration. Depending on the route of administration, theactive compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditionswhich may inactivate the compound. For examinpfordinistrationofagents, byother than parenteral administration, it may be desirable to coat the agent with., or co-administer the agent with, a material to prevent its inactivation. An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzymc inhibitors or in an appropriate carrier such as liposomes- Pharmaceutiticaly acceptablediuentsinclude saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes anyimmune stimulating compoundsuchasinteron. Adjuvants contemplated herein include resorcinols, non Ionic surfactants such as polyoxyethylene oleyl etherand n-hexadecy polyethylene ether, Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEEP) and trasylol Liposomes include water-in-oil-in-water emulsions as well as conventional liposones (Sterna et al (1984) JNeuroiimmw/ 7:27), Theagentxmay also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols,and mixtures thereof, and in oils. Underordinaryconditions of storage and use, these preparations maycontain a preservative to prevent the growth of iicroorgansis. Pharmaceutical compositions of agentssuitable for injectableuse include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extenporaneous preparation of sterile injectable solutions or dispersion, In all cases the composition will preferably be sterile and must be fluid to the extent that easy syringeabilityexists. It will preferably bestableunder the conditions ofmanufactureand storage and preserved against the contaminating action ofnicroorgamsn such as bacteria and fungi, The carrier can be a solvent or dispersionmedium containing, tor example, water, ethanol, polynl(for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as leithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surtfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal,and the like. In many cases, it is preferable to include isotonic agentsfor example, sugars, polvalcohols such as mniatol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearateand gelatin. Sterile injectablesolutions can be prepared by iiorporating an agent of the invention (e.g. an antibody, peptide, fusion protein or small molecule) in the required amount in an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparationare vacuum drying and freeze-draig which Yields a powder of the agent plus any additional desired ingredient from a previously sterile-filtered solution thereof When the agent is suitably protected, as described above, the protein can be orally administered, for example, with aninert diluent oran assimilable edible carrier, As used herein"pharmaceuticallyacceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and an-tifungal agents- isotonie and absorption delaying agents, and the like. The use of such media and agentsfor pharmaceutically active substances is well knownin theart.FExceptinsofarasanyconventionalmediaoragentis incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage. "Dosage unit form", as used herein, refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect inassociatin with the required pharmaceutical carrier. The specification for the dosage uiit forms of the invention are dictated by, and directly dependent on, (a) the unique characteristicsOf the active compoundand the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art ofcompounding suchan active compound for the treatment ofsensitivity in individuals.

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In one embodiment. an agent of the invention is an antibody, As defined herein, a therapeutically effective amount of antibody ( e, an effective dosage) ranges from about 0,001 to 30 mg/kg body weight, preferably about 001 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferbly about 1 to 10 ng/k,2 to 9 n/3 to 8 g/kg, 4 to 7 mg/kg, or 5 to 6migkg body weight, The skilled artisan will appreciate that certain Factrsmayinfluencethedosage required to effectively treat asubject, including but not limited to the severity ofthe disease or disorder, previous treatments, thegeneral health and/orage of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can iclude a series of treatments, In a preferred example, a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about I to 10 weeks, preferably between 2 to 8 weeks, miore preferably between about 3 to7 weeks, and.even more preferably for about 4, 5, or 6 weeks- It will also beappreciated that the effective dosage of antibody used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results of diagnostic assays, addition, anantibody ofthe inventon can also be administered in combination therapy with, e.g., chmotherapeutic agents, hormones, antangiogens, radiolabelled, compounds, or with surgerycrotherapy, and/or radiotherapy. An antibody of the invention canalso be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post conventional therapy. For example, the antibody can be administered With a therapeutically effective dose of chemotherapeutic agent. In another embodiment, the antibody can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment ofvarious cancers. The dosing regimenand dosages of these aforementioned chemotherapeutic drugs thatare therapeutically effective will depend on the particular immune disorder, e, Hodgkin lymphoma, being treated. the extent of the disease and other factors familiar to the physician of skill in the art and canhe determined bytie physician. In addition, the agents of the invention described herein can beadministered using nanoparticle-based composition and delivery methods well known to the skilled artisan. For example, nanoparticle-based delivery for improved nucleic acid(ag, smail RNAs) therapeutics are well known in the art (Expert Opinion onBiological lherapv 7:1811 1822).

Exemplification This invention is further illustrated by the following exampleswhich should not be construed as limiting.

Example I: HPV-Mediated PD-41 Splice Variants are Associated withHead, Neck, and LungCancers and Other Cancers Figure I shows a schematic of a mutationallandscape of head and neck cancers analyzed by The Cancer Genome Atlas (TCGA) project. Sequencing results of:279tumors using whole-exome hybrid capture identified genes displaying significant enrichment for mutation in this dataset. The start indicates samples with evidence of HPV infection, Each column represents one tumor and the different types of tumors are shown by the different marks. Also listed are the number of mutations per sample, thepercent of samples with mutationsineachofthelistedgenes and. whether these genes have been reported in the COSMIC (Sanger Cancer Gene Census) database. Figure 2 shows data in the same format as forFigure 1, except that the data is drawn from an independent cohort of HPV- (left half) and HPV+ (right half) head and neck cancers in which 700 cancer-related geneswere sequenced. Figure 2 furthershows amplified (middle box) or deleted genes (bottom box) Figure 3 shows a representation of sites of -PV integration in the host genome in head and neck cancers analyzed as part of the TCGA project described in Figure 1. The circle shows the human chromosomes around the perimeter and. the green lines show sites of integration. Figures 4 and 5 show sequencing reads for the CD274 (PD-L I) gene in a squamous head and neck cancertumor from the larynx (CV-5433) with a detected HPV integration in the PD-Li gene. On theright panel is seen a white space in the center of the reads which corresponds to where HPV is detected (see left panel). The site of integrationsspans positions 5,464,244 to 5,464.509 on chromosome 9 of the Human Genome Reference Consortium Human Build 37 (GRCh37/hg]9) assembly publicly available as GenBaik Assembly ID: GCA 000001405.1 and RefSeq Assembly ID: iCF 000001405,13 dated February 27, 2009. Figure 5 shows a zoomed in view of Figure 4 according toa log scale.

Figure 6 showsa consolidation of the sequencing read data of Figures 4 and 5 into a schematic showing the IPV integration within the PD-L gene of tumor CV-5433. Figure 7 shows predicted protein products following IPV integration in tunor CV 5433. One protein isa short form of PD-L4(soluble 1) made of 227 aminoacids (plusa stop codon) instead of the full-length, membrane-bound form having 290 amino acids(Le, the nucleic acid ncodes froi 5' to 3' the following domains: Exon1 (L-region), Exon 2 (IgV-like-domain), Exon 3 (IgC-like-domain), Exon 4 (the connecting-relon, transmembraneegionand the start of the intracytoplasmic region), and Exons 5 and 6 (intracytop)1asmic-regon) The identified short form of PD-LJ. results in loss of the transmembiinandintracellulardomaistouceneratcasolobe/secretedpolypeptide.In additionanother shortfonn of PDL I (soluble 2) was identified and confirmed via RNA sequencing as being identical to the soluble I form, but further containing an additional C terminal sequence (Figure 7) that isnot contained in the wild-type,fall-length, menbrane bound PD-LI form, Figure 8 shows expression of each exon of full-length, membrane-bound PD-L1 on a log scale from the CV-5433 sample and demonstrates a dramatic drop in exons following exon 4 which is the site of HPV integration. Figures 9-10 show transcript Varants of PD-L Iexpressd by head, neck., and hng cancers. Figure 9 shows that samples fromthe TCOA headand neck (left panel) and lung cancer righth pacl) projects display a diversity of splicing in PD~LIindicating that many tumors produce soluble PD-LL Figure 10 shows the data from Figure 9 for head and neck cancers in a different manner. Each head and neck tumor is represented asa circle and the tumors are ordered left to rightaccording to total PD-LI expression Tumors which fivor the short form are lower on the y-axis and normal samples are shown as square boxes. The sample with the[HPV integration is labeledas the index case and is a clear outlier in terms of high PD-L i expression favoring the soluble form. Figure I Ishows that, in addition to head and neck cancer (-INSC), RNA sequencing analyses generated by TCGA (The Cancer Genome Atlas) using computiational methods to detect PD-Li short forms identified expression of such forms in bladder cancer (BLCA), breast cancer (BRCA), glioblastorna (GBM), kidney cancer (KIRC),acute myelogeious leukemia (LANL) lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian cancer (OV), and also summarized in a pan-cancer graph. Tumors are shown in dark gray and normal samples, when viable, are shown in light gray. The y-axis indicates total PD-LI expressionand the x-axis indicates the fraction of short PD-L IAmplification of PD-Li is shown by the intensity of the circle, The results indicate that there isa spectrum of PD-LI expression across many cancer types with many tumors displaying evidence of at least some production of the shortform, typically consistent with the soluble 2 form having the C-terminal sequence, VIPGNlNTSIKIL SSThe soluble 2 frm sequences were identified by analysis of an Index case of head and neck cancer from The Cancer Genome Atlas project (TCGA-CV 5443) in which we identified integration of HPV in the PDL (CD274) gene using the Pathseq algorithm as described above. Assembly of PDLI. transcripts from RNA sequencing data from this tumor using Cufflinks demonstrated a proportion of transcripts with evidence of sequence beyond the normal exon boundary. Manual assembly and inspection of these transcripts revealed the presence of sequence corresponding to the downstream intron as well as alternative poladenylation of truncated PDL I nRNA leading to the translation of the amino acids "G N L N V S J K IC L T L S P1S " which are not part of fill-length, wild-typePDI. Evidence of similar transcripts was identified in other primarytumors and cancer derived cell lines, Figure 12 shows similar results using similar analyses as those conducted in Figure 11 using the Cancer Cell Line Encyclopedia dataset. Since the TCGA samples shown in Figure 11 are bulk tumors which contain both tumorand normal stroma, the source of soluble/short PD couldbeither the tumor ornormal immune cells. However, Fiure 12 denonstratcs that cancer cells grown without any stroma produce short PD-L L The circle at approximately -12 on the y-axisand 038 onthex-axisand within the circlesatthe very top row of circles on the y-axis is the index head and neck case for comparison.

Example2: Soluble PD-L IIsoforms Associated with Head, Neck, and Lung Cancers and Other Cancers are Immunologically Active Figures 13-14 show Western blot results 293T cells transfected with expression vectors encoding the full-length or short PD-L i forms, Figure 13 demonstrates that full length PD-LI is only foud. in the cells (left)and the short form is only found in themedia (right). thereby confirming that the short form is soluble. Figure 14 demonstrates that the shortformofPD-Ll wasproduced andaffinity purified. Specificdlly, nucle.icacid molecues encodin full4enth PD-LI were purchased. from Origne and PCR was used to introducean carly termination codon. PCR was also used toadd 3 HA epitope tags. The short and long forms were cloned into the expression vector pCDNA3 and also pBabe Puro and pMSCV puro. Cell lines expressing short and long PD-L forms were created by introducing the PD-LI transgene by transient transfection (293T cells) or retroviral infection (K562 and Ba/F3 cells). HA.I 1Clone 16B12 monoclonal antibody was used to detect the 1A epitope tag and the ab58810 polyclonal antibody to detect PD-L (Abcam). Figure 15 shows the detection of short PDL 1 in culture media obtained from cell lines predicted to make higher amounts of using immunoblot analyses (eg, the RKO colon cancer cell lines, the BDCI acute myelogenous leukemia cell fine, and the CAL62 thyroid anaplastic carcinoma cell line). This was confirmed by ELISA with cells cngineredto make the short form (293T, 293T PDL1 long (e acwild-type, full-length, membrane-bound PD-.J)_PD-Ll short),as ell as cancer cell lines predicted to produce thesoluble form (eg., the RKO and CAL62 cell lines). For recombinant production of full-length and soluble PDL, a c-terminal HA tag was attached and an ani-HA antibody (Thermo 26183) was used for detection. For endogenous PDLl, the 368A5A4 antibody was used for WesternblottingandtheELISA29E12BIantibodywasusedforELSAassays(courtesy of Gordon Freeman's laboratory; see also Brown et al (2003)L Immunol 170:1257-1266 Figure 15 further shows the distribution of PD-L Iin cells engineered to make thewild-type orshortforms- which indicates that the short form is present in the media Figure 16 shows the results of T cell viability assays using T cells obtained from twoindependent donorsand incubated in the presence of vehicle or either of the two short PDLl fomnis (i.e, PD-L IS - Soluble 1 or PD-Li S2 - Soluble 2) or media from cells engineered to make wild-type PD- Iin which most should have been retained in the cell. Briefly, T cells were isolated from peripheral blood from healthy blood donors at the Dana Farber Cancer institute using theauto-MACS system. Tcells were cultured in RPMI media andstimulated with CD3 and CD28 conjugated antibody beads for 48 hours. Recombinant soluble PDL I(both forms) produced via HA-tag affinyit purification from transiently transfected HEK293T cells was then as added at 10 micrograms/mil and cells were cultured for an additional 48 hours. Cell viability was measured by propidium iodide exclusion usinga flow cytometer, As a control HA-afinity purification was performed from the supernatant of 293T cells expressing full-length PDL1 using exactly the same conditions as for the soluble form. Etaposide at 10 micronolar was used as a positive control forcell death. For conditions in which blocking antibodies were used, the blocking antibodies were addedat 10 micrograms per nil and the following antibodies from the Freeman laboratory were utlizd: anti-human PD-I129E 2A3 and anti-hunan PD-IEH 12 (see, Brown el at (2003) Anunol 170:A257- 1266; Rodig eat a (2003) Eu, Ikmnunol 3117-3126; Cai et al. (2004) Cell hnnuol 230,89-9; Dorfiman et a (2,06) Amer J SurPathol 30:802-810; Day el al (2006)NaNure 443:350-3)54) Flgure 16 shows reduction MT cell viability by approximatey 10-20% with soluble PD-Lc treatment, but further shows that this effect can be blunted by addition of anti-PD1 oranti-PDLi antibodies, Figure 17-48 shows that the short form of PD-L can differentially kill proliferating Tells. Figure 17 shows the results ofT cells obtained from a healthy donor and stimulated to proliferate and then treated with either a) the short form of PD-L (undiluted,"high inpuf'), b) the short fon of PD-L (diluted, "low input"), c) the long formof PD-L 1, or d) the chemotherapeutic topoisomerase inhibitor, etoposide. The treated T cells then underwent flow cvyometry analysis to determine the proportion of live cells. CD8 staining is shown on thex-axis and. cell viability is shown on the Y-axis. The resultsshow thai thc short form of PD-L killsT cells similar to toposide and likely CD4 and not CD8 cells Figure 18 shows the results of the experiment described in Figure17exceptthattheTcells were not stimulated to proliferate.

Incorporation by Reference The contents of all references, patentapplications, patents; and published patent applications, as well as the Figures and theSequence Listing, cited throughout this application are hereby incorporated byreference.

Equivalents Those skilled in the artwill recognize, or beable toascertain using no more than routine experimentation, many equivalents to the specific enbodinents of the invention described herein. Such equivalents are intended to be encompassed by thefollowing claims.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

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Claims (19)

1. What is claimed: 1. A method of determining whether a subject is afflicted with, or at risk for developing a head, neck, or lung cancer, or whether said subject would benefit from modulating PD-1 and/or PD-Li levels, the method comprising: a) using a sample from the subject; b) determining the copy number, level of expression, or level of activity of one or more soluble PD-Li isoforms, or a fragment thereof in the subject sample; c) determining the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms, or a fragment thereof in a control; and d) comparing the copy number, level of expression, or level of activity of said one or more soluble PD-Li isoforms detected in steps b) and c); wherein a significant increase in the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms indicates that (i) the subject is afflicted with the head, neck, or lung cancer, or (ii) is at risk for developing the head, neck, or lung cancer, or (iii) said subject would benefit from modulating PD-Li and/or PD-Li levels, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
2. 2. A method for monitoring the progression of a head, neck, or lung cancer in a subject, the method comprising: a) detecting in a subject sample at a first point in time the copy number, level of expression, or level of activity of one or more soluble PD-Li isoforms, or a fragment thereof; b) repeating step a) at a subsequent point in time; and c) comparing the copy number, level of expression, or level of activity of said one or more soluble PD-Li isoforms detected in steps a) and b) to monitor the progression of the head, neck, or lung cancer, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
3. 3. The method of claim 2, wherein an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample at a first point in time relative to the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample at a subsequent point in time indicates progression of the head, neck, or lung cancer; or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample at a first point in time relative to the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample at a subsequent point in time indicates a lack of significant progression of the head, neck, or lung cancer.
4. 4. The method of claim 2 or 3 wherein between the first point in time and the subsequent point in time, the subject has undergone treatment to modulate PD-i and/or PD-Li levels.
5. 5. A method for stratifying subjects afflicted with a head, neck, or lung cancer according to predicted clinical outcome of treatment with one or more modulators of PD-1 and/or PD-Li levels, the method comprising: a) determining the copy number, level of expression, or level of activity of one or more one or more soluble PD-Li isoforms, or a fragment thereof in a subject sample; b) determining the copy number, level of expression, or level of activity of the one or more one or more soluble PD-Li isoforms, or a fragment thereof in a control sample; and c) comparing the copy number, level of expression, or level of activity of said one or more soluble PD-Li isoforms detected in steps a) and b); wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample relative to the normal copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the control sample predicts the clinical outcome of the patient to treatment with one or more modulators of PD-1 and/or PD-Li levels, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO: 13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
6. 6. The method of claim 5, wherein the predicted clinical outcome is (a) cellular growth, (b) cellular proliferation, or (c) survival time resulting from treatment with one or more modulators of PD-1 and/or PD-Li levels; and/or wherein an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample compared to the control sample predicts that the subject has a poor clinical outcome; or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the subject sample compared to the control sample predicts that the subject has a favorable clinical outcome.
7. 7. The method of claim I or 2, further comprising the use of a therapeutic agent that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkers selected from PD-1, PD-Li, and soluble isoforms of PD Li.
8. 8. A method of determining the efficacy of a test compound for inhibiting a head, neck, or lung cancer in a subject, the method comprising: a) determining the copy number, level of expression, or level of activity of one or more soluble PD-Li isoforms, or a fragment thereof in a first sample obtained from the subject and exposed to the test compound; b) determining the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms, or a fragment thereof in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and c) comparing the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the first and second samples, wherein a significantly modulated copy number, level of expression, or level of activity of the soluble PD-Li isoforms, relative to the second sample, is an indication that the test compound is efficacious for inhibiting the head, neck, or lung cancer in the subject, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15, preferably wherein the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject.
9. 9. A method of determining the efficacy of a therapy for inhibiting a head, neck, or lung cancer in a subject, the method comprising: a) determining the copy number, level of expression, or level of activity of one or more soluble PD-Li isoforms, or a fragment thereof in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject; b) determining the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms, or a fragment thereof in a second sample obtained from the subject following provision of the portion of the therapy; and c) comparing the copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the first and second samples, wherein a significantly decreased copy number, level of expression, or level of activity of the one or more soluble PD-Li isoforms in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the head, neck, or lung cancer in the subject, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
10. 10. A method for identifying a compound which inhibits a head, neck, or lung cancer, the method comprising: a) contacting one or more soluble PD-Li isoforms or a fragment thereof with a test compound; and b) determining the effect of the test compound on the copy number, or level of expression of the one or more soluble PD-Li isoforms to thereby identify a compound which inhibits the head, neck, or lung cancer, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
11. 11. The method of claim 10, wherein the one or more soluble PD-Li isoforms is expressed in a cell, preferably wherein said cells are isolated from an animal model of a head, neck, or lung cancer, and/or wherein said cells are from a subject afflicted with a head, neck, or lung cancer.
12. 12. A pharmaceutical composition comprising a non-natural antisense polynucleotide having a modified phosphate backbone that specifically binds to a polynucleotide of one or more soluble PD-Li isoforms or a fragment thereof when used for treating a head, neck, or lung cancer in a pharmaceutically acceptable carrier, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
13. 13. The composition when used of claim 12, wherein the antisense polynucleotide further comprises an expression vector.
14. 14. A pharmaceutical composition when used of claim 12 for treating a head, neck, or lung cancer in a human subject.
15. 15. The method of any one of claims 1, 3, and 4, wherein the control is determined from a non-cancerous sample from the subject or member of the same species to which the subject belongs, or wherein the sample consists of or comprises body fluid, cells, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject, preferably wherein the body fluid is selected from group consisting of amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, and vomit.
16. 16. The method of any one of claims 1-11, wherein the copy number is assessed by microarray, quantitative PCR (qPCR), high-throughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH), or wherein the expression level of the one or more soluble PD-Li isoforms is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the soluble PD-Li isoform or a portion of said polynucleotide molecule, preferably wherein the polynucleotide molecule is a mRNA, cDNA, or functional variants or fragments thereof and, optionally, wherein the step of detecting further comprises amplifying the polynucleotide molecule, or wherein the expression level of the one or more soluble PD-Li isoforms is assessed by annealing a nucleic acid probe with the sample of the polynucleotide encoding the one or more soluble PD-Li isoforms or a portion of said polynucleotide molecule under stringent hybridization conditions, or wherein the expression level of the soluble PD-Li isoform is assessed by detecting the presence in the samples of a protein of the soluble PD-Li isoform, a polypeptide, or protein fragment thereof comprising said protein, preferably wherein the presence of said protein, polypeptide or protein fragment thereof is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment thereof, preferably wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment, or wherein the activity level of the soluble PD-Li isoform is assessed by determining the magnitude of modulation of the activity or expression level of downstream targets of the one or more soluble PD-Li isoforms.
17. 17. The method of any one of claims 7, 8, 10, and 11, wherein the agent or test compound modulates PD-1, PD-Li, and or soluble PD-Li levels, or wherein the agent or test compound is an antibody against soluble PD-Li, or wherein the agent or test compound is a small molecule inhibitor of soluble PD-LI, or wherein the agent or test compound is an anti-PD-Li inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shRNA, siRNA, aptamer, ribozyme, and dominant-negative protein binding partner.
18. 18. Use of a non-natural antisense polynucleotide having a modified phosphate backbone that specifically binds to a polynucleotide of one or more soluble PD-LI isoforms or a fragment thereof in the manufacture of a medicament for treating a head, neck, or lung cancer, wherein the one or more soluble PD-Li isoforms comprise an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least

    80% identical to SEQ ID NO:13 or 15; or comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15 or an amino acid sequence that is at least 80% identical to SEQ ID NO:13 or 15.
19. 19. The method or composition when used, or use of any one of claims 1-18, wherein the head or neck cancer is squamous cell carcinomas of the head and neck (SCCHN) or associated with human papillomavirus infection, or wherein the lung cancer is small-cell lung carcinoma (SCLC) or non-small-cell lung carcinoma (NSCLC).